peerj_json_files/PeerJ_Json_104.json

{
    "v1_Abstract": "Our previously presented method for high throughput computational screening of mutant activity (Hediger et al., [1]) is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). Using an appropriate cutoff criterion for the computed barriers, the qualitative activity of 15 out of 22 mutants is correctly predicted. The method identifies four of the six most active mutants with \u22653-fold wild type activity and seven out of the eight least active mutants with \u22640.5-fold wild type activity. The method is further used to screen all sterically possible (386) double-, tripleand quadruple-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified.",
    "v1_col_introduction": "introduction  : In industry, one frequently tries to modify an enzyme in order to enhance its functionality in a certain way [2\u20136]. From an application point of view, one of the most interesting questions is how to modify an enzyme such that its activity is enhanced compared to wild type or such that a new kind of activity is introduced into the enzyme [7,8]. It can therefore be of considerable relevance to have a method available which efficiently allows to a priori discriminate between promising candidates for experimental study and mutants which can be excluded from the study.\nPeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013)\nR ev ie w in g M an\n2 Numerous methods are currently being proposed and developed for the description of enzyme activities, the theoretical background of which ranges from phenomenological and bioinformatics based approaches [9\u201313] to quantum mechanics based ab initio descriptions [14\u201322]. However one can expect that methods which are highly demanding in terms of set-up efforts and computational time are less likely to be employed in industrial contexts where qualitative or semi-quantitative conclusions can be of sufficient use in the beginning and planning phase of a wet-lab study. Few approaches, while taking into account a number of approximations and limitations in accuracy, aim at being used in parallel or prior to experimental work [23,24] and are not designed to be used for high throughput fashion. Hediger et al. have recently published a computational method for high throughput computational screening of mutant activity [1] and in this paper we benchmark the method against experimentally measured amidase activity for mutants of Candida antarctica lipase B (CalB) and apply the method to identify additional promising mutants.",
    "v2_Abstract": "Our previously presented method for high throughput computational screening of mutant activity (Hediger et al., [1]) is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). Using an appropriate cutoff criterion for the computed barriers, the qualitative activity of 15 out of 22 mutants is correctly predicted. The method identifies four of the six most active mutants with \u22653-fold wild type activity and seven out of the eight least active mutants with \u22640.5-fold wild type activity. The method is further used to screen all sterically possible (386) double-, tripleand quadruple-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified.",
    "v2_col_introduction": "introduction  : In industry, one frequently tries to modify an enzyme in order to enhance its functionality in a certain way [2\u20136]. From an application point of view, one of the most interesting questions is how to modify an enzyme such that its activity is enhanced compared to wild type or such that a new kind of activity is introduced into the enzyme [7,8]. It can therefore be of considerable relevance to have a method available which efficiently allows to a priori discriminate between promising candidates for experimental study and mutants which can be excluded from the study.\nPeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013)\nR ev ie w in g M an\n2 Numerous methods are currently being proposed and developed for the description of enzyme activities, the theoretical background of which ranges from phenomenological and bioinformatics based approaches [9\u201313] to quantum mechanics based ab initio descriptions [14\u201322]. However one can expect that methods which are highly demanding in terms of set-up efforts and computational time are less likely to be employed in industrial contexts where qualitative or semi-quantitative conclusions can be of sufficient use in the beginning and planning phase of a wet-lab study. Few approaches, while taking into account a number of approximations and limitations in accuracy, aim at being used in parallel or prior to experimental work [23,24] and are not designed to be used for high throughput fashion. Hediger et al. have recently published a computational method for high throughput computational screening of mutant activity [1] and in this paper we benchmark the method against experimentally measured amidase activity for mutants of Candida antarctica lipase B (CalB) and apply the method to identify additional promising mutants.",
    "v1_text": "results and discussion : methods : We introduce the experimental set-up and the methodology for comparing experimental and computational data. We describe a benchmarking and a combinatorial study of CalB mutant activity. Experimentally, variants of Candida Antarctica lipase B (CalB) were either produced in Pichia pastoris with C-terminal His6-tag for subsequent affinity purification or expressed in Aspergillus oryzae without terminal tag followed by a three-step purification procedure. It is generally accepted that in serine protease like enzymes, the formation of the tetrahedral intermediate (TI, Fig. 1) is rate determining [25\u201328] and throughout this work we assume that a lower barrier for this reaction correlates to increased overall activity of the enzyme. The substrate used throughout this study is N-benzyl-2-chloroacetamide. The organisms used for expression of the individual variants are indicated in Table 1. conclusions : Our previously presented method for high throughput computational screening of mutant activity [1] is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 10 Experimentally, amidase activity is successfully introduced in 12 mutants, the highest activity is determined to be 11.2 -fold over the wild type activity. Using an appropriate cutoff criterion for the computed barriers, the qualitative activity of 15 out of 22 mutants is correctly predicted. It is noted that the correlation is best for mutants with largest activity difference compared to wild type (both positive and negative). For example the method identifies four of the six most active mutants with \u22653-fold wild type activity. Similarly, the method identifies seven out of the eight least active mutants with \u22640.5-fold wild type activity. Thus validated, the computational method is used to screen all sterically possible (386) double-, tripleand quadrupole-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified. These mutants have so far not been tested experimentally and are thus offered as scientifically testable predictions. Interestingly, we observe that single mutants that are predicted to have low activity appear to have high activity in combination with other mutants. This is illustrated in specific analysis of effects of mutations of two different positions (104 and 189). tables : Table 1. Experimental overall activities and calculated reaction barriers of Set S. Activity factors +1/-1 indicate increased/decreased overall activity. Ao and Pp indicating expression in organisms (Org.) Aspergillus oryzae or Pichia pastoris, respectively. The cutoff to distinguish higher and lower activity mutants is defined as 12.5 kcal/mol, see text. Species Experimental Calculated Activity [*WT] ActivityFactor Barriers [kcal/mol] ActivityFactor Org. G39A-T103G-W104F-L278A 11.2 1 13.9 -1 Ao G39A-L278A 7.0 1 11.3 1 Pp G39A-W104F 4.2 1 10.6 1 Ao G39A-T103G-L278A 3.8 1 7.3 1 Ao G39A-W104F-L278A 3.6 1 11.8 1 Pp T103G 3.0 1 13.6 -1 Ao G39A-W104F-I189Y-L278A 2.9 1 10.9 1 Pp G39A 2.8 1 11.2 1 Ao L278A 2.5 1 12.8 -1 Ao W104F 2.0 1 12.0 1 Ao G39A-T103G-W104Q-L278A 1.9 1 12.8 -1 Ao G39A-T103G-W104F-D223G-L278A 1.5 1 11.3 1 Pp G39A-T103G 0.8 -1 7.5 1 Ao G39A-T42A-T103G-W104F-L278A 0.7 -1 10.4 1 Pp I189H 0.5 -1 12.9 -1 Pp G39A-I189G-L278A 0.4 -1 10.7 1 Pp G41S 0.3 -1 13.4 -1 Pp I189G 0.2 -1 18.9 -1 Pp G39A-T103G-W104F-I189H-D223G-L278A 0.1 -1 13.7 -1 Pp G39A-T103G-W104F-I189H-L278A-A282G-I285A-V286A 0.1 -1 12.9 -1 Pp A132N 0.0 -1 12.5 -1 Pp P38H 0.0 -1 12.5 -1 Pp WT 1.0 -- 7.5 -- PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 20 Table 2. Point mutations. The term active site refers to residues with potential direct Van der Waals contact to the substrate. The term first shell/second shell refers to residues which are adjecent to an active site/first shell residue. Target Mutations Type Description P38 H Second shell (H neutral) G39 A First shell G41 S First shell T42 A Second shell T103 G First shell W104 F, Q, Y Active site A132 N First shell A141 N, Q Active site I189 A, G, H, N, Y Active site (G including additional water, H neutral) D223 G First shell (Increase of charge by +1) L278 A Active site A282 G Active site I285 A Active site V286 A First shell PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 21 Table 3. Side chains used for generation of combinatorial set L. i and gi indicate the position in the back bone and the number of mutations at that position, respectively. Mutation i gi G39A 39 1 T103G 103 1 W104{F, Q, Y} 104 3 A141{N, Q} 141 2 I189{A, G, H, N, Y} 189 5 L278A 278 1 PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 22 Table 4. Combinatorial study details. From the possible mutants, the combinations containing the pair A141N/Q-I189Y, the mutants with inconclusive barriers and the mutants with barriers >19.0 kcal/mol are subtracted to give the number of mutants in set L. \u201cOnly Set L\u201d indicates the number of mutants uniquely present in set L and not in set S. Order Possible Containing Inconclusive Barrier >19.0 Set L Only A141N/Q-I189Y barrier [kcal/mol] Set L Single 13 0 0 0 13 7 Double 64 2 4 8 50 47 Triple 154 12 21 20 101 98 Four-fold 193 24 36 19 114 111 Total 424 38 61 47 278 263 PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 23 Table 5. Selection of mutants from set L with lowest barriers. Mutation Barrier[kcal/mol] G39A-T103G-I189Y 5.7 G39A-I189Y 6.2 G39A-A141Q-I189G-L278A 6.3 G39A-A141N-L278A 7.6 G39A-A141N 7.7 G39A-A141N-I189H-L278A 8.3 G39A-W104F-A141Q-I189A 8.3 G39A-A141Q-I189N 9.1 G39A-A141N-I189N 9.3 G39A-T103G-W104Y-A141N 9.8 G39A-W104Y-I189Y 9.8 G39A-A141N-I189N-L278A 10.1 G39A-W104F-A141N 10.1 G39A-I189H-L278A 10.2 G39A-A141N-I189A-L278A 10.2 W104Y-I189H 10.4 G39A-T103G-W104F-I189Y 10.4 G39A-A141Q-I189A-L278A 10.4 G39A-T103G-A141Q-I189H 10.4 G39A-T103G-I189A-L278A 10.5 PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t Figure 1 Reaction scheme for the formation of TI. Nucleophilic attack by O\u03b3 of S105 on carbonyl carbon C20 of substrate. R1: -CH2-Cl, R2: -CH2-C5H6. PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t Figure 2 Position of point mutations. A. Overlay of mutations W104[F, Q, Y]. B: Overlay of mutations A141[N, Q]. C: Overlay of mutations of I189[A, G, H, N, Y]. D: Mutations P38H, G39A, G41S, T42A, T103G, A132N, L278A, A282G, I285A, V286A. Substrate shown in magenta. PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t Figure 3 Comparison of experimental and computed activities. 1/-1 correspond to increased/decreased overall activity, respectively. Prediction rate is 15/22 (68%). PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t Figure 4 Barrier scatter plot of set S. 22 mutants; The cutoff value cS is discussed in the text. PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t Figure 5 Barrier scatter plots of set L. In both panels, the labels indicate mutants containing the labeled and possibly additional mutations up to the indicated order. \"OTHER\" indicates a mutant not containing any of the labeled mutations or of higher than 4. order. A: Mutations of W104. B: Mutations of I189. PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t computational details : The computational method used to estimate the reaction barriers of the CalB mutants has been described in detail earlier [1] and is only summarized here. As described previously [1], in order to make the method computationally feasible, relatively approximate treatments of the wave function, structural model, dynamics and reaction path are used. Given this and the automated setup of calculations, some inaccurate results will be unavoidable. However, the intent of the method is similar to experimental high throughput screens of enzyme activity where, for example, negative results may result from issues unrelated to intrinsic activity of the enzyme such as imperfections in the activity assay, low expression yield, protein aggregation, etc. Just like its experimental counterpart our technique is intended to identify potentially interesting mutants for further study. The reaction barriers are estimated computationally by preparing molecular model structures [1] (consisting of around 840 atoms) of the enzyme substrate complex (ES) and the tetrahedral intermediate (TI) inbetween which linear interpolation is carried out to generate structures of the enzyme on the reaction path. Such adiabatic mapping is the most common way to estimate barriers in QM/MM studies of enzymatic reaction mechanisms. The resulting barriers tend to be in good agreement with experiment, which indicates that this is a reasonable approximation (see for example [30] and [16]). The geometry of each interpolation frame is optimized while keeping the distance between the nucleophilic carbon C20 of the substrate and O\u03b3 of serine 105 (Fig. 1) fixed at a specific value di = dini \u2212 i(dini \u2212 dfin)/10, where dini and dfin are the distances between C 20 and O\u03b3 in the ES complex and TI, respectively (in A\u030a, 10 PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 5 being the number of interpolation frames and i the interpolation frame index). In geometry optimization calculations, the gradient convergence criteria is set to 0.5 kcal/(molA\u030a) and a linear scaling implementation of the PM6 method (MOZYME [31]) together with a NDDO cutoff of 15 A\u030a is applied. The energy profile of the reaction barrier at the PM6 level of theory [32] is subsequently mapped out by carrying out conventional SCF calculations of each optimized interpolation frame. All calculations are carried out using the MOPAC suite of programs [33, 34]. The molecular models are based on the crystal structure of the CalB enzyme with PDB identifier 1LBS [35]. In order to prevent significant rearrangement of hydrogen bonding network of surface residues during the optimization, a number of additional structural constraints are applied in the geometry optimizations, i.e. the residues S50, P133, Q156, L277 and P280 are kept fixed. These (surface) residues are observed to rearrange and form new hydrogen bonds in optimizations when no constraints are applied. Omitting the constraints leads to unconclusive barrier shapes containing many irregular minima along the reaction coordinate which do not permit to readily define a reaction barrier. For the analysis, the reaction barrier is defined by the difference between the highest energy point on the reaction profile and the energy corresponding to the enzyme substrate complex. From our calculations (PM6//MOZYME in vacuum), we estimate the wild type (WT) barrier to be 7.5 kcal/mol. Experimentally, specific activity of hydrolysis is determined. Given first order kinetics, saturation of the enzyme with substrate (usual for industrial application) and fast binding and product release, the catalytic rate constant kcat is directly proportional to the specific activity under the assumption that the amount of active enzyme remains constant. This therefore allows the catalytic rate constant kcat and, hence, the barrier height to be compared to the improvement factors reported in the results section. The approximations used here in relating the barrier height on the potential energy surface to kcat have been discussed previously [1]. It is noted that using one CPU per interpolation frame on the reaction barrier, the complete barrier of one mutant can be computed with 10 CPUs usually within less than 12 hours of wall clock time (for a molecular model of the size used in this study). Given a set of molecular models of the enzyme, and 100 available CPUs, it is possible to screen around 1000 mutants within one week. generation of calb variants without his-tags : Variants of CalB carrying the CalB signal peptide were generated at the DNA level using QuickChange mutagenesis on the corresponding gene residing in a dual E.coli/Aspergillus Pichia pastoris expression vector. The PCR was performed with proofreading DNA polymerase (New England Biolabs, NEB). To PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 3 remove parent templates, they were methylated in vitro prior to PCR with CpG methyltransferase (from NEB) and digested in vivo after transformation of competent E.coli DH5 \u03b1 cells (TaKaRa) according to the instructions from the manufacturer. Plasmid DNA was isolated from transformed E.coli strains, and sequenced to verify the presence of the desired substitutions. Confirmed plasmid variants were used to transform an Aspergillus oryzae strain that is negative in pyrG (orotidine-5\u2019-phosphate decarboxylase), proteases pepC (aserine protease homologous to yscB), alp (an alkaline protease) NpI (a neutral metalloprotease I) to avoid degradation of the lipase variants during and after fermentation. The transformed Aspergillus strains were fermented as submerged culture in shake flasks and the lipase variants secreted into the fermentation medium. After the fermentation, the lipase variants were purified from the sterile filtered fermentation medium in a 3 step procedure with 1) hydrophobic interaction chromatography on decylamine-agarose, 2) buffer exchange by gel filtration and 3) ion exchange chromatography with cation exchange on SP-sepharose at pH 4.5. The lipase variant solutions were stored frozen. Variants of CalB carrying the CalB signal peptide and C-terminal His-tags were generated at the DNA level using SOE-PCR and inserted into a dual E.coli/Pichia pastoris expression vector using In-fusion cloning (ClonTech). The SOE-PCR was performed with Phusion DNA polymerase (NEB) and template DNA of the CalB gene. The cloned plasmids were transformed in competent E.coli DH5 \u03b1 cells (TaKaRa). Plasmid DNA was isolated from transformed E.coli strains, and sequenced to verify the presence of the desired substitutions. Confirmed plasmid variants were used to transform a Pichia pastoris strain that is Mut(s), Suc(+), His(-). The transformed Pichia strains were fermented as submerged culture in deep well plates and secretion of the lipase variants into the fermentation medium was induced by addition of methanol. After the fermentation, the lipase variants were purified from the cleared supernants using a standard His-tag purification protocol (Qiagen) and buffer-exhanged into 50 mM phosphate buffer, pH 7.0, using Amicon Ultra centrifugal filter devices with a 10 kDa cutoff (Merck Millipore). activity measurement : Amidase activity of CalB variants was determined in a two-step fluorimetric assay previously described by Henke et al [29]. First, enzymatic hydrolysis of N-benzyl-2-chloroacetamide was performed in 96-well microtiter plates in 200 \u00b5L phosphate-buffered aqueous solution pH 7.0 including 10% organic co-solvent PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 4 (THF or DMSO). Reactions containing 5 mM amide substrate, 0.3-3 \u00b5M enzyme, and 12 \u00b5g/mL BSA were incubated for 18-20 h at 37\u25e6C in a shaker incubator. In a second step, 50 \u00b5L of a 20 mM 4- nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-Cl) solution in 1-hexanol was added and the reaction of NBD-Cl with benzylamine formed during amide hydrolysis proceeded under identical reaction conditions for another hour. Fluorescence of the final reaction product was determined with excitation at 485 nm and measured emission at 538 nm. Calibration of the amide hydrolysis reaction was performed on each assay plate with benzylamine covering a concentration range between 0.05 and 5 mM. All enzymatic activities were corrected for non-enzymatic background reaction determined under identical conditions without enzyme present. combination mutants : The molecular model of the enzyme and the positions of the point mutations in the enzyme are illustrated in Fig. 2. The point mutations are listed in Table 2. Two sets of mutants are introduced in this section. PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 6 A benchmarking set S and a combinatorial set L, the definitions of which are provided in the following. The point mutations are selected based on different design principles. These are either introduction of structural rearrangements in the active site to change the binding site properties of the active site (residues P38, G39, G41, T42, T103) [2], introduction of space to accomodate the substrate (W104, L278, A282, I285, V286), introduction of dipolar interactions between the enzyme and the substrate (A132, A141, I189) [36] or reduction of polarity in the active site (D223). Of course different heuristic considerations will apply for other enzymes when selecting the single mutations for combinatorial study. The mutants of the benchmarking study are collected in a small set S (22 mutants, Table 1). For the combinatorial study, out of the above we select six residues (G39, T103, W104, A141, I189, L278) which, it is assumed, contribute strongest to increased activity and define the mutations at each position as listed in Table 3. Given the position i and the number of mutations at each position gi, in general the upper limit for the number of mutants M in a combinatorial study can be calculated by writing a sum term for each type (i.e. \u201corder\u201d) of combination mutant, i.e. single, double, . . . , such that M = \u2211 i gi \ufe38 \ufe37\ufe37 \ufe38 Single (o=1) + \u2211 i,j j>i gi \u00b7 gj \ufe38 \ufe37\ufe37 \ufe38 Double (o=2) + \u2211 i,j,k k>j>i gi \u00b7 gj \u00b7 gk \ufe38 \ufe37\ufe37 \ufe38 Triple (o=3) + . . . (1) where each sum term consists of ( N o ) individual terms (N and o being the number of positions which can be mutated and the order of the mutant, respectively). By this scheme, considering the mutations listed in Table 3, hypothetically 424 (= 13 + 64 + 154 + 193) single to four-fold mutants can be constructed. This number is reduced by applying the restriction that out of the 424 hypothetically possible mutants, 0 single, 2 double, 12 triple and 24 four-fold combination mutants including the pair A141N/Q-I189Y are discarded because in the molecular modeling, these side chains could not be allocated spatially in the same mutant. We further note that 15 out of these remaining 386 mutants (Table 3) are present also in the benchmarking set S and thus the combinatorial study consists of 371 unique mutants. A detailed documentation of the number of screened residues in the combinatorial study is provided in Table 4. Prior to analysis, the reaction barriers of the combination mutants are inspected visually and mutants with irregularly shaped barriers, i.e. consisting of multiple peaks of similar height along the reaction coordinate, are discarded. This step is done simply because the calculations yield inconclusive results, PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 7 so the most conservative choice is to consider it a non-promising candidate for a more active variant. Generating plots of the profiles is completely automated and visual inspection can easily be done for hundreds of mutants. Furthermore, out of the mutants with regular reaction barrier shapes, we discard those mutants with barriers >19.0 kcal/mol (i.e. the largest calculated barrier from set S). Following these selection criteria, 61 mutants are discarded because of inconclusive barrier shapes and 47 mutants because the barrier is higher than 19 kcal/mol (a distribution of reaction barriers is shown in Fig. S1). After these filtering steps, 278 mutants remain in the combinatorial study which we collect in the large set L (out of which 15 are in set S). An overview on the distribution of reaction barriers for the mutants from set L is provided Fig. S2 of the supporting information. We note that in set S, all barriers appear regular in shape and no mutant contains the A141N/Q and I189Y pair. set s: calibration of the accuracy : The correspondance of the computed barriers from set S with the experimental assay is shown in Fig. 3. The exact data is reported in Table 1. A scatterplot of calculated reaction barriers is presented in Fig. 4. We note that in set S, the highest experimentally observed activity is around 11 times the wild type activity (G39A-T103G-W104F-L278A, Table 1), while roughly ten mutants show no increased activity. In total, six mutants show 3-fold or higher wild type activity. In the calculations, only one mutant is observed to have a lower barrier than the wild type (7.3 kcal/mol, G39A-T103G-L278A) and the highest observed barrier is 18.9 kcal/mol (I189G). Given the approximations introduced to make the method sufficiently efficient, it is noted that the intent of the method is not a quantitative ranking of the reaction barriers, but to identify promising mutants for, and to eliminate non-promising mutants from, experimental consideration. Therefore only qualitative changes in overall activity are considered, which are represented by the activity factors (+1/-1). We categorize the experimentally observed activities and the predicted reaction barriers as follows. From experiment, a mutant with activity of 1.2 (0.8) times the wild type activity or higher (lower) is considered PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 8 as improving (degrading). Correspondingly, the computed difference in reaction barrier height between a mutant and the wild type is expressed in qualitative terms. For the comparison with the experimental activity assay, we define a barrier cutoff cS = 12.5 kcal/mol to distinguish between potentially improving and degrading mutants in set S. The value of 12.5 kcal/mol is chosen such as to maximize the agreement with experiment, which is 68%, i.e. using a smaller or larger value for the cutoff will decrease this value. A mutant with a predicted barrier \u2265cS (12.5 kcal/mol) is considered to likely have decreased activity compared to the wild type while mutants with reaction barriers <cS are considered likely having increased activity. We note that defining the cutoff is done purely for a post hoc comparison of experimental and computed data. When using the computed barriers to identify promising experimental mutants, one simply chooses the N mutants with the lowest barriers, where N is the number of mutants affordable to do experimentally (e.g. 20 in the discussion of set L). Based on this approach, qualitative activity of 15 out of 22 mutants is correctly predicted. It is noted that the correlation is best for mutants with largest activity difference compared to wild type (both positive or negative). For example the method identifies four of the six most active mutants with \u22653-fold wild type activity. Similarly, the method identifies seven out of the eight least active mutants with \u22640.5-fold wild type activity. For mutants with only small differences in activity compared to wild type, the predictions are less accurate. Set L: Large scale screening study Set L is screened to identify new mutants for which increased activity is predicted. The 20 mutants with the lowest barriers are suggested as candidates for further experimental study in Table 5. The distributions of reaction barriers, resolved by mutations at positions 104 and 189, are shown in Figs. 5A and B. In set L, three new mutants are identified with barriers lower than the predicted wild type barrier. Out of the 20 mutants suggested in Table 5, three are double mutants, seven are three-fold and ten are four-fold mutants. No single mutants where found for which increased activity compared to wild type is predicted. All mutants except one contain the G39A mutation, five contain the T103G mutation, six contain a mutation of W104, 13 contain a mutation of A141, 16 contain a mutation of I189 and eight contain the L278A mutation. From this observation it is likely that mutations of G39, A141 and I189 will likely contribute to an increased activity of the mutant and should thus be included in future experimental activity assays. PeerJ reviewing PDF | (v2013:07:643:1:0:NEW 6 Aug 2013) R ev ie w in g M an us cr ip t 9 Set L is further analysed in terms of the effect of the mutations at the positions 104 and 189. For the mutations of W104, we note that single mutations which give rise to relatively high barriers (W104Q, W104Y, Fig. 5A) can have significantly lower barriers in combination with other mutations. For example, out of the sixty mutants with lowest barriers (Fig. S3), 33 contain a mutation of W104 out of which 17 are suggested to be W104F, while 14 are suggested to be W104Y (two contain W104Q). The mutation of I189 is analysed in a similar way. In set L, five different mutations of this residue are screened (Table 3). The single mutant with the lowest barrier is I189Y and the two mutants with the lowest predicted barrier contain this mutation as well (Table 5). Similarly to above, higher order mutants containing I189A, I189G, I189H or I189N are predicted to have considerably lower barriers than the corresponding single mutants, Fig. 5B. Particularly, out of the mutants listed in Table 5, three contain the I189A, one contains I189G mutation, four contain the I189H mutation and three contain the I189N mutation. As a special case we highlight that the single mutant I189G has one of the highest calculated barriers (18.9 kcal/mol, Table 1), however the four-fold mutant G39A-A141Q-I189G-L278A has one of the lowest barriers (6.3 kcal/mol, Table 5). Interestingly, the mutant G39A-A141Q-L278A has an intermediate barrier (10.9 kcal/mol). It would appear that I189G as a single mutant is counterproductive (high computed barrier) but lowers the barrier of G39A-A141Q-L278A. This observation is further supported by the observation that the I189G mutation is in spatial proximity to A141Q. While it is difficult to quantify the interaction, it is likely that in the mutant, the rather large side chain of A141Q is better accommodated in the active site and can better interact with the substrate. Observations as these should be kept in mind when selecting the single mutants to be considered when preparing higher order mutants.",
    "v2_text": "results and discussion : figure legends : ES TS TI PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 15 ! \" # $ !\"#$% &'(% )*+'& ,*$(-%./&./0./1./23 %*4*-1./53 6*+4-7./5./23 8'$0 9\"\"'& &4*: )4\"% %\"$\"& ,\"$;% <\"$=% %*'\"1 Figure 2. Positions of point mutations. A: Overlay of mutations W104[F, Q, Y]. B: Overlay of mutations of A141[N, Q]. C: Overlay of mutations of I189[A, G, H, N, Y]. D: Mutations P38H, G39A, G41S, T42A, T103G, A132N, L278A, A282G, I285A and V286A. Substrate shown in magenta. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 16 PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 17 1 2 3 4 5 6 7 8 5 10 15 20 M ut at io n O rd er (1 -8 ) Reaction Barrier [kcal/mol] (calculated) PM6//MOZYME cS = 12.5 kcal/mol Set S Figure 4. Barrier scatter plot of set S. 22 mutants; The cutoff value cS is discussed in the text. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 18 PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 19 methods : We introduce the experimental set-up and the methodology for comparing experimental and computational data. We describe a benchmarking and a combinatorial study of CalB mutant activity. Experimentally, variants of Candida Antarctica lipase B (CalB) were either produced in Pichia pastoris with C-terminal His6-tag for subsequent affinity purification or expressed in Aspergillus oryzae without terminal tag followed by a three-step purification procedure. It is generally accepted that in serine protease like enzymes, the formation of the tetrahedral intermediate (TI, Fig. 1) is rate determining [25\u201328] and throughout this work we assume that a lower barrier for this reaction correlates to increased overall activity of the enzyme. The substrate used throughout this study is N-benzyl-2-chloroacetamide. The organisms used for expression of the individual variants are indicated in Table 1. 2 novozymes a/s, krogshoejvej 36, dk-2880 bagsv\u00e6rd, denmark : \u2217 Corresponding Author, Email: jhjensen@chem.ku.dk conclusions : Our previously presented method for high throughput computational screening of mutant activity [1] is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). Experimentally, amidase activity is successfully introduced in 12 mutants, the highest activity is determined to be 11.2 -fold over the wild type activity. Using an appropriate cutoff criterion for the computed barriers, the qualitative activity of 15 out of 22 mutants is correctly predicted. It is noted that the correlation is best for mutants with largest activity PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 10 difference compared to wild type (both positive and negative). For example the method identifies four of the six most active mutants with \u22653-fold wild type activity. Similarly, the method identifies seven out of the eight least active mutants with \u22640.5-fold wild type activity. Thus validated, the computational method is used to screen all sterically possible (386) double-, tripleand quadrupole-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified. These mutants have so far not been tested experimentally and are thus offered as scientifically testable predictions. Interestingly, we observe that single mutants that are predicted to have low activity appear to have high activity in combination with other mutants. This is illustrated in specific analysis of effects of mutations of two different positions (104 and 189). 1 department of chemistry, university of copenhagen, universitetsparken 5, dk-2100 : tables : Table 1. Experimental overall activities and calculated reaction barriers of Set S. Category (Cat.) +1/-1 indicates increased/decreased overall activity. Category definition is discussed in text. Ao and Pp indicating expression in organisms (Org.) Aspergillus oryzae or Pichia pastoris, respectively. Species Exp. Activity [*WT] Cat. Calc. Barriers [kcal/mol] Cat. Org. G39A-T103G-W104F-L278A 11.2 1 13.9 -1 Ao G39A-L278A 7.0 1 11.3 1 Pp G39A-W104F 4.2 1 10.6 1 Ao G39A-T103G-L278A 3.8 1 7.3 1 Ao G39A-W104F-L278A 3.6 1 11.8 1 Pp T103G 3.0 1 13.6 -1 Ao G39A-W104F-I189Y-L278A 2.9 1 10.9 1 Pp G39A 2.8 1 11.2 1 Ao L278A 2.5 1 12.8 -1 Ao W104F 2.0 1 12.0 1 Ao G39A-T103G-W104Q-L278A 1.9 1 12.8 -1 Ao G39A-T103G-W104F-D223G-L278A 1.5 1 11.3 1 Pp G39A-T103G 0.8 -1 7.5 1 Ao G39A-T42A-T103G-W104F-L278A 0.7 -1 10.4 1 Pp I189H 0.5 -1 12.9 -1 Pp G39A-I189G-L278A 0.4 -1 10.7 1 Pp G41S 0.3 -1 13.4 -1 Pp I189G 0.2 -1 18.9 -1 Pp G39A-T103G-W104F-I189H-D223G-L278A 0.1 -1 13.7 -1 Pp G39A-T103G-W104F-I189H-L278A-A282G-I285A-V286A 0.1 -1 12.9 -1 Pp A132N 0.0 -1 12.5 -1 Pp P38H 0.0 -1 12.5 -1 Pp WT 1.0 -- 7.5 -- PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 20 Table 2. Point mutations. The term active site refers to residues with potential direct Van der Waals contact to the substrate. The term first shell/second shell refers to residues which are adjecent to an active site/first shell residue. Target Mutations Type Description P38 H Second shell (H neutral) G39 A First shell G41 S First shell T42 A Second shell T103 G First shell W104 F, Q, Y Active site A132 N First shell A141 N, Q Active site I189 A, G, H, N, Y Active site (G including additional water, H neutral) D223 G First shell (Increase of charge by +1) L278 A Active site A282 G Active site I285 A Active site V286 A First shell PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 21 Table 3. Side chains used for generation of combinatorial set L. i and gi indicate the position in the back bone and the number of mutations at that position, respectively. Mutation i gi G39A 39 1 T103G 103 1 W104{F, Q, Y} 104 3 A141{N, Q} 141 2 I189{A, G, H, N, Y} 189 5 L278A 278 1 PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 22 Table 4. Combinatorial study details. From the possible mutants, the combinations containing the pair A141N/Q-I189Y, the mutants with inconclusive barriers and the mutants with barriers >19.0 kcal/mol are subtracted to give the number of mutants in set L. \u201cOnly Set L\u201d indicates the number of mutants uniquely present in set L and not in set S. Order Possible Containing Inconclusive Barrier >19.0 Set L Only A141N/Q-I189Y barrier [kcal/mol] Set L Single 13 0 0 0 13 7 Double 64 2 4 8 50 47 Triple 154 12 21 20 101 98 Four-fold 193 24 36 19 114 111 Total 424 38 61 47 278 263 PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 23 Table 5. Selection of mutants from set L with lowest barriers. Mutation Barrier[kcal/mol] G39A-T103G-I189Y 5.7 G39A-I189Y 6.2 G39A-A141Q-I189G-L278A 6.3 G39A-A141N-L278A 7.6 G39A-A141N 7.7 G39A-A141N-I189H-L278A 8.3 G39A-W104F-A141Q-I189A 8.3 G39A-A141Q-I189N 9.1 G39A-A141N-I189N 9.3 G39A-T103G-W104Y-A141N 9.8 G39A-W104Y-I189Y 9.8 G39A-A141N-I189N-L278A 10.1 G39A-W104F-A141N 10.1 G39A-I189H-L278A 10.2 G39A-A141N-I189A-L278A 10.2 W104Y-I189H 10.4 G39A-T103G-W104F-I189Y 10.4 G39A-A141Q-I189A-L278A 10.4 G39A-T103G-A141Q-I189H 10.4 G39A-T103G-I189A-L278A 10.5 PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t ESI-1 computational details : The computational method used to estimate the reaction barriers of the CalB mutants has been described in detail earlier [1] and is only summarized here. As described previously [1], in order to make the method computationally feasible, relatively approximate treatments of the wave function, structural model, dynamics and reaction path are used. Given this and the automated setup of calculations, some inaccurate results will be unavoidable. However, the intent of the method is similar to experimental high throughput screens of enzyme activity where, for example, negative results may result from issues unrelated to intrinsic activity of the enzyme such as imperfections in the activity assay, low expression yield, protein aggregation, etc. Just like its experimental counterpart our technique is intended to identify potentially interesting mutants for further study. The reaction barriers are estimated computationally by preparing molecular model structures [1] (consisting of around 840 atoms) of the enzyme substrate complex (ES) and the tetrahedral intermediate (TI) inbetween which linear interpolation is carried out to generate structures of the enzyme on the reaction path. Such adiabatic mapping is the most common way to estimate barriers in QM/MM studies of enzymatic reaction mechanisms. The resulting barriers tend to be in good agreement with experiment, which indicates that this is a reasonable approximation (see for example [30] and [16]). The geometry of each interpolation frame is optimized while keeping the distance between the nucleophilic carbon C20 of the substrate and O\u03b3 of serine 105 (Fig. 1) fixed at a specific value di = dini \u2212 i(dini \u2212 dfin)/10, where dini and dfin are the distances between C 20 and O\u03b3 in the ES complex and TI, respectively (in A\u030a, 10 PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 5 being the number of interpolation frames and i the interpolation frame index). In geometry optimization calculations, the gradient convergence criteria is set to 0.5 kcal/(molA\u030a) and a linear scaling implementation of the PM6 method (MOZYME [31]) together with a NDDO cutoff of 15 A\u030a is applied. The energy profile of the reaction barrier at the PM6 level of theory [32] is subsequently mapped out by carrying out conventional SCF calculations of each optimized interpolation frame. All calculations are carried out using the MOPAC suite of programs [33, 34]. The molecular models are based on the crystal structure of the CalB enzyme with PDB identifier 1LBS [35]. In order to prevent significant rearrangement of hydrogen bonding network of surface residues during the optimization, a number of additional structural constraints are applied in the geometry optimizations, i.e. the residues S50, P133, Q156, L277 and P280 are kept fixed. These (surface) residues are observed to rearrange and form new hydrogen bonds in optimizations when no constraints are applied. Omitting the constraints leads to unconclusive barrier shapes containing many irregular minima along the reaction coordinate which do not permit to readily define a reaction barrier. For the analysis, the reaction barrier is defined by the difference between the highest energy point on the reaction profile and the energy corresponding to the enzyme substrate complex. From our calculations (PM6//MOZYME in vacuum), we estimate the wild type (WT) barrier to be 7.5 kcal/mol. Experimentally, specific activity of hydrolysis is determined. Given first order kinetics, saturation of the enzyme with substrate (usual for industrial application) and fast binding and product release, the catalytic rate constant kcat is directly proportional to the specific activity under the assumption that the amount of active enzyme remains constant. This therefore allows the catalytic rate constant kcat and, hence, the barrier height to be compared to the improvement factors reported in the results section. The approximations used here in relating the barrier height on the potential energy surface to kcat have been discussed previously [1]. It is noted that using one CPU per interpolation frame on the reaction barrier, the complete barrier of one mutant can be computed with 10 CPUs usually within less than 12 hours of wall clock time (for a molecular model of the size used in this study). Given a set of molecular models of the enzyme, and 100 available CPUs, it is possible to screen around 1000 mutants within one week. generation of calb variants without his-tags : Variants of CalB carrying the CalB signal peptide were generated at the DNA level using QuickChange mutagenesis on the corresponding gene residing in a dual E.coli/Aspergillus Pichia pastoris expression vector. The PCR was performed with proofreading DNA polymerase (New England Biolabs, NEB). To PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 3 remove parent templates, they were methylated in vitro prior to PCR with CpG methyltransferase (from NEB) and digested in vivo after transformation of competent E.coli DH5 \u03b1 cells (TaKaRa) according to the instructions from the manufacturer. Plasmid DNA was isolated from transformed E.coli strains, and sequenced to verify the presence of the desired substitutions. Confirmed plasmid variants were used to transform an Aspergillus oryzae strain that is negative in pyrG (orotidine-5\u2019-phosphate decarboxylase), proteases pepC (aserine protease homologous to yscB), alp (an alkaline protease) NpI (a neutral metalloprotease I) to avoid degradation of the lipase variants during and after fermentation. The transformed Aspergillus strains were fermented as submerged culture in shake flasks and the lipase variants secreted into the fermentation medium. After the fermentation, the lipase variants were purified from the sterile filtered fermentation medium in a 3 step procedure with 1) hydrophobic interaction chromatography on decylamine-agarose, 2) buffer exchange by gel filtration and 3) ion exchange chromatography with cation exchange on SP-sepharose at pH 4.5. The lipase variant solutions were stored frozen. Variants of CalB carrying the CalB signal peptide and C-terminal His-tags were generated at the DNA level using SOE-PCR and inserted into a dual E.coli/Pichia pastoris expression vector using In-fusion cloning (ClonTech). The SOE-PCR was performed with Phusion DNA polymerase (NEB) and template DNA of the CalB gene. The cloned plasmids were transformed in competent E.coli DH5 \u03b1 cells (TaKaRa). Plasmid DNA was isolated from transformed E.coli strains, and sequenced to verify the presence of the desired substitutions. Confirmed plasmid variants were used to transform a Pichia pastoris strain that is Mut(s), Suc(+), His(-). The transformed Pichia strains were fermented as submerged culture in deep well plates and secretion of the lipase variants into the fermentation medium was induced by addition of methanol. After the fermentation, the lipase variants were purified from the cleared supernants using a standard His-tag purification protocol (Qiagen) and buffer-exhanged into 50 mM phosphate buffer, pH 7.0, using Amicon Ultra centrifugal filter devices with a 10 kDa cutoff (Merck Millipore). activity measurement : Amidase activity of CalB variants was determined in a two-step fluorimetric assay previously described by Henke et al [29]. First, enzymatic hydrolysis of N-benzyl-2-chloroacetamide was performed in 96-well microtiter plates in 200 \u00b5L phosphate-buffered aqueous solution pH 7.0 including 10% organic co-solvent PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 4 (THF or DMSO). Reactions containing 5 mM amide substrate, 0.3-3 \u00b5M enzyme, and 12 \u00b5g/mL BSA were incubated for 18-20 h at 37\u25e6C in a shaker incubator. In a second step, 50 \u00b5L of a 20 mM 4- nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-Cl) solution in 1-hexanol was added and the reaction of NBD-Cl with benzylamine formed during amide hydrolysis proceeded under identical reaction conditions for another hour. Fluorescence of the final reaction product was determined with excitation at 485 nm and measured emission at 538 nm. Calibration of the amide hydrolysis reaction was performed on each assay plate with benzylamine covering a concentration range between 0.05 and 5 mM. All enzymatic activities were corrected for non-enzymatic background reaction determined under identical conditions without enzyme present. combination mutants : The molecular model of the enzyme and the positions of the point mutations in the enzyme are illustrated in Fig. 2. The point mutations are listed in Table 2. Two sets of mutants are introduced in this section. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 6 A benchmarking set S and a combinatorial set L, the definitions of which are provided in the following. The point mutations are selected based on different design principles. These are either introduction of structural rearrangements in the active site to change the binding site properties of the active site (residues P38, G39, G41, T42, T103) [2], introduction of space to accomodate the substrate (W104, L278, A282, I285, V286), introduction of dipolar interactions between the enzyme and the substrate (A132, A141, I189) [36] or reduction of polarity in the active site (D223). Of course different heuristic considerations will apply for other enzymes when selecting the single mutations for combinatorial study. The mutants of the benchmarking study are collected in a small set S (22 mutants, Table 1). For the combinatorial study, out of the above we select six residues (G39, T103, W104, A141, I189, L278) which, it is assumed, contribute strongest to increased activity and define the mutations at each position as listed in Table 3. Given the position i and the number of mutations at each position gi, in general the upper limit for the number of mutants M in a combinatorial study can be calculated by writing a sum term for each type (i.e. \u201corder\u201d) of combination mutant, i.e. single, double, . . . , such that M = \u2211 i gi \ufe38 \ufe37\ufe37 \ufe38 Single (o=1) + \u2211 i,j j>i gi \u00b7 gj \ufe38 \ufe37\ufe37 \ufe38 Double (o=2) + \u2211 i,j,k k>j>i gi \u00b7 gj \u00b7 gk \ufe38 \ufe37\ufe37 \ufe38 Triple (o=3) + . . . (1) where each sum term consists of ( N o ) individual terms (N and o being the number of positions which can be mutated and the order of the mutant, respectively). By this scheme, considering the mutations listed in Table 3, hypothetically 424 (= 13 + 64 + 154 + 193) single to four-fold mutants can be constructed. This number is reduced by applying the restriction that out of the 424 hypothetically possible mutants, 0 single, 2 double, 12 triple and 24 four-fold combination mutants including the pair A141N/Q-I189Y are discarded because in the molecular modeling, these side chains could not be allocated spatially in the same mutant. We further note that 15 out of these remaining 386 mutants (Table 3) are present also in the benchmarking set S and thus the combinatorial study consists of 371 unique mutants. A detailed documentation of the number of screened residues in the combinatorial study is provided in Table 4. Prior to analysis, the reaction barriers of the combination mutants are inspected visually and mutants with irregularly shaped barriers, i.e. consisting of multiple peaks of similar height along the reaction coordinate, are discarded. This step is done simply because the calculations yield inconclusive results, PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 7 so the most conservative choice is to consider it a non-promising candidate for a more active variant. Generating plots of the profiles is completely automated and visual inspection can easily be done for hundreds of mutants. Furthermore, out of the mutants with regular reaction barrier shapes, we discard those mutants with barriers >19.0 kcal/mol (i.e. the largest calculated barrier from set S). Following these selection criteria, 61 mutants are discarded because of inconclusive barrier shapes and 47 mutants because the barrier is higher than 19 kcal/mol (a distribution of reaction barriers is shown in Fig. S1). After these filtering steps, 278 mutants remain in the combinatorial study which we collect in the large set L (out of which 15 are in set S). An overview on the distribution of reaction barriers for the mutants from set L is provided Fig. S2 of the supporting information. We note that in set S, all barriers appear regular in shape and no mutant contains the A141N/Q and I189Y pair. set s : The correspondance of the computed barriers from set S with the experimental assay is shown in Fig. 3. The exact data is reported in Table 1. A scatterplot of calculated reaction barriers is presented in Fig. 4. We note that in set S, the highest experimentally observed activity is around 11 times the wild type activity (G39A-T103G-W104F-L278A, Table 1), while roughly ten mutants show no increased activity. In total, six mutants show 3-fold or higher wild type activity. In the calculations, only one mutant is observed to have a lower barrier than the wild type (7.3 kcal/mol, G39A-T103G-L278A) and the highest observed barrier is 18.9 kcal/mol (I189G). Given the approximations introduced to make the method sufficiently efficient, it is noted that the intent of the method is not a quantitative ranking of the reaction barriers, but to identify promising mutants for, and to eliminate non-promising mutants from, experimental consideration. Therefore only qualitative changes in overall activity are considered. We categorize the experimentally observed activities and the predicted reaction barriers as follows. From experiment, a mutant with activity of 1.2 (0.8) times the wild type activity or higher (lower) is considered PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 8 as improving (degrading). Correspondingly, the computed difference in reaction barrier height between a mutant and the wild type is expressed in qualitative terms. For the comparison with the experimental activity assay, we define a barrier cutoff cS = 12.5 kcal/mol to distinguish between potentially improving and degrading mutants in set S. A mutant with a predicted barrier \u2265cS (12.5 kcal/mol) is considered to likely have decreased activity compared to the wild type while mutants with reaction barriers <cS are considered likely having increased activity. We note that defining the cutoff is done purely for a post hoc comparison of experimental and computed data. When using the computed barriers to identify promising experimental mutants, one simply chooses the N mutants with the lowest barriers, where N is the number of mutants affordable to do experimentally (e.g. 20 in the discussion of set L). Based on this approach, qualitative activity of 15 out of 22 mutants is correctly predicted. It is noted that the correlation is best for mutants with largest activity difference compared to wild type (both positive or negative). For example the method identifies four of the six most active mutants with \u22653-fold wild type activity. Similarly, the method identifies seven out of the eight least active mutants with \u22640.5-fold wild type activity. For mutants with only small differences in activity compared to wild type, the predictions are less accurate. Set L is screened to identify new mutants for which increased activity is predicted. The 20 mutants with the lowest barriers are suggested as candidates for further experimental study in Table 5. The distributions of reaction barriers, resolved by mutations at positions 104 and 189, are shown in Figs. 5A and B. In set L, three new mutants are identified with barriers lower than the predicted wild type barrier. Out of the 20 mutants suggested in Table 5, three are double mutants, seven are three-fold and ten are four-fold mutants. No single mutants where found for which increased activity compared to wild type is predicted. All mutants except one contain the G39A mutation, five contain the T103G mutation, six contain a mutation of W104, 13 contain a mutation of A141, 16 contain a mutation of I189 and eight contain the L278A mutation. From this observation it is likely that mutations of G39, A141 and I189 will likely contribute to an increased activity of the mutant and should thus be included in future experimental activity assays. Set L is further analysed in terms of the effect of the mutations at the positions 104 and 189. For the PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t 9 mutations of W104, we note that single mutations which give rise to relatively high barriers (W104Q, W104Y, Fig. 5A) can have significantly lower barriers in combination with other mutations. For example, out of the sixty mutants with lowest barriers (Fig. S3), 33 contain a mutation of W104 out of which 17 are suggested to be W104F, while 14 are suggested to be W104Y (two contain W104Q). The mutation of I189 is analysed in a similar way. In set L, five different mutations of this residue are screened (Table 3). The single mutant with the lowest barrier is I189Y and the two mutants with the lowest predicted barrier contain this mutation as well (Table 5). Similarly to above, higher order mutants containing I189A, I189G, I189H or I189N are predicted to have considerably lower barriers than the corresponding single mutants, Fig. 5B. Particularly, out of the mutants listed in Table 5, three contain the I189A, one contains I189G mutation, four contain the I189H mutation and three contain the I189N mutation. As a special case we highlight that the single mutant I189G has one of the highest calculated barriers (18.9 kcal/mol, Table 1), however the four-fold mutant G39A-A141Q-I189G-L278A has one of the lowest barriers (6.3 kcal/mol, Table 5). Interestingly, the mutant G39A-A141Q-L278A has an intermediate barrier (10.9 kcal/mol). It would appear that I189G as a single mutant is counterproductive (high computed barrier) but lowers the barrier of G39A-A141Q-L278A. Observations as these should be kept in mind when selecting the single mutants to be considered when preparing higher order mutants. electronic supplementary information : In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB Martin R. Hediger1, Luca De Vico1, Julie B. Rannes2, Christian Ja\u0308ckel2, Werner Besenmatter2, Allan Svendsen2, Jan H. Jensen1\u2217 copenhagen, denmark : s1 barrier scatter plots : ! \" # $ % ! ! ! \" ! # ! $ ! % & ! & \" & # & $ & % \" ! \" \" \" # \" $ \" % ' ! ' \" ' # ' $ ( # !( !# &( &# \"( )*+,-./0/1234-54+ 234-5416) 7 - .. *0 .1 89 , - :; < = :> Figure S1. Discarded mutants with barriers > 19.0 kcal/mol. Single mutants: 0; Double mutants: 8; Triple mutants: 20; Four-fold mutants: 19; Total: 47. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t ESI-2 ! \" ! # $ $ $ % & ' ( & # ) # * ' ( * ! * \" \" # % $ % % ! ) ' ! ! & ! $ ) ! $ * ! & ( ! ( ! ! ( \" ! # # ! ' $ ! ' % ! * ' ! \" & ! % ) ! % * $ ) ( $ ! ! $ ! \" $ $ # $ & $ $ & % $ ( ' $ # & $ ' ) $ ' * $ * ( ) $ ( ' \" !) !$ !( !' !\" +,--./-012310/4151674,840 974,841:; < 8 / -= > 1? @ A , BC 6 2 BD Figure S2. Combination mutants from set L. Single: 13; Double mutants: 50; Triple mutants: 101; Four-fold mutants: 114: Total: 278. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t ESI-3 ! \" # $ % & ' ( ) !* !! !\" !# !$ !% !& !' !( !) \"* \"! \"\" \"# \"$ \"% \"& \"' \"( \") #* #! #\" ## #$ #% #& #' #( #) $* $! $\" $# $$ $% $& $' $( $) %* %! %\" %# %$ %% %& %' %( %) &* * \" $ & ( !* !\" !$ +,-./-0123-4156270-18.993790 :!*$; :!*$< :!*$= >-479 +,-./-1?@ A . 99 37 91 BC D . 5E F 6 5G Figure S3. Low-barrier mutants. 33 out of 60 contain a mutation of W104 (W104F: 17, W104Y: 14, W104Q: 2). PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t Figure 1 Reaction scheme for the formation of TI. Nucleophilic attack by O\u03b3 of S105 on carbonyl carbon C20 of substrate. R1: -CH2-Cl, R2: -CH2-C5H6. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t Figure 2 Position of point mutations. A. Overlay of mutations W104[F, Q, Y]. B: Overlay of mutations A141[N, Q]. C: Overlay of mutations of I189[A, G, H, N, Y]. D: Mutations P38H, G39A, G41S, T42A, T103G, A132N, L278A, A282G, I285A, V286A. Substrate shown in magenta. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t Figure 3 Comparison of experimental and computed activities. 1/-1 correspond to increased/decreased overall activity, respectively. Prediction rate is 15/22 (68%). PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t Figure 4 Barrier scatter plot of set S. 22 mutants; The cutoff value cS is discussed in the text. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t Figure 5 Barrier scatter plots of set L. In both panels, the labels indicate mutants containing the labeled and possibly additional mutations up to the indicated order. \"OTHER\" indicates a mutant not containing any of the labeled mutations or of higher than 4. order. A: Mutations of W104. B: Mutations of I189. PeerJ reviewing PDF | (v2013:07:643:0:0:NEW 16 Jul 2013) R ev ie w in g M an us cr ip t",
    "url": "https://peerj.com/articles/147/reviews/",
    "review_1": "Fred Muench \u00b7 Aug 6, 2013 \u00b7 Academic Editor\nACCEPT\nThank you for making the changes to the introduction - the paper will be useful to help understand this research area. The paper can be accepted but please make the following changes:\n1. Please copy edit the entire manuscript. There were several typos\n2. Line 24: At present little is known about.... I would start the paragraph with - line 27 - We wished to conduct a systematic review -- first. Then At present... (Switch order). That will help transition the reader - then leading to why you did this preliminary paper.\n\nCongratulations.",
    "review_2": "Fred Muench \u00b7 Mar 19, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nThank you for making the edits to the document. It enhances the manuscript. My main concern at this point is the introduction. It is now extremely short and the paragraphs do not necessarily flow after items were deleted without modifying the text - and new text was not included. I apologize if this was not clear in the previous edit but is important for the best possible manuscript. I believe the introduction should at minimum:\n\n1- have an additional paragraph on how health videos are being used presently across health disciplines and the information/intervention they are attempting to convey to consumers. For example, it is post-op care instructions, general health advice, etc. Please give examples.Even though this is a description of reviews this will help orient the reader who may not be experts.\n\n2- Your main question is the following: \"In reviews and systematic reviews of health-related YouTube videos, what are common reporting practices, exemplary methods and methodological challenges.\"\n\nAfter the above inserted paragraph, please write an additional paragraph on the problem or challenge you are trying to address with your paper -- what discrepancies exist, why conduct this review aside from the prevalence of these videos for example. More information on the importance of this review is also important to orient the reader.\n\nIn general the background should be clearer and set the reader up for your methods.\n\nBased on all your research, this should be a minor revision. Please contact me with questions. Fred",
    "review_3": "Fred Muench \u00b7 Jan 17, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nAs with the two reviewers, I believe this article has merit and should be accepted with revisions. Understanding how to evaluate consumer health videos is an important area of study which will only grow . Please be sure to copy edit the manuscript as there are several errors. I have three broad based suggestions (some corresponding to reviewers) that may improve the manuscript for the reader and several smaller ones. First, the introduction appears to focus more of the background of how you came to the decision to study health videos by focusing on infant pain which is never mentioned again. It would be helpful to discuss in more detail about who is creating these videos, more on why this is an important area of study and most importantly to bring it back to your conclusion and goals \u2013 what are the current accepted systematic review methods (lines 316) and why we need to modify these current methods for videos and create a new method. In the method section, please merge how you searched into one paragraph (e.g. add 61-63 into the paragraph). Please define \u201csnowball techniques\u201d in more detail. Line 138 appears to be missing some text. The review provided so much important information as presented in the table and was a well done descriptive study. As with reviewer two, it would be nice to see you expand the discussion and conclusions based on your knowledge and findings to include \u2013 what are the most important areas that researchers cover when conducting reviews of Web-based health videos beyond the descriptive table of what you covered, why it is important and include a sample rubric, and with your knowledge, it would be very interesting to include a brief paragraph on what individuals developing videos should take into account - though this is clearly a separate paper and just a suggestion to enhance the paper for a larger audience. Looking forward to seeing the revised manuscript.",
    "review_4": "Reviewer 1 \u00b7 Jan 16, 2013\nBasic reporting\nWhile I am unfamiliar with the journal, the research question seems well-identified, contained, and sized.\nExperimental design\nThe research question is clear. This study is strictly descriptive, which is fine for addressing the authors' research question. Methods were well-described.\nValidity of the findings\nIt seems likely that these findings are valid, inasmuch as the methods for the present review are well-described.\nAdditional comments\nThis paper is a useful description of \u201creviews of reviews\u201d of YouTube videos related to health. It specifies many dimensions of review reporting, and in some cases makes recommendations (e.g., for stopping rules). As information sources grow in channel types, attempts to summarize it will need to develop standards for reporting, and papers such as this one will help to make this happen.\nIt is interesting that some institutions required IRB review for the studies and some did not; were there differences between the two categories that account for this?\nOne issue that puzzles me a bit has to do with \u201csnowballing\u201d to include videos that appear as recommendations alongside the \u201cindex\u201d video. It seems that using this procedure would result in non-replicable results (if replicability is even a relevant issue here), and would interfere with systematicity in the search. A description of how this strategy could be made more systematic would be helpful.\nI would request that the authors attend carefully to their use of the terms \u201creview\u201d and \u201cstudy\u201d. Sometimes reviews are called studies, and this is confusing: for example, in line 260 where the study that is referred to is the pain-during-vaccination one that initiated this project.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"A systematic review of methods for studying consumer health YouTube videos, with implications for systematic reviews (v0.1)\". PeerJ https://doi.org/10.7287/peerj.147v0.1/reviews/1",
    "pdf_1": "https://peerj.com/articles/147v0.3/submission",
    "pdf_2": "https://peerj.com/articles/147v0.2/submission",
    "review_5": "Reviewer 2 \u00b7 Dec 29, 2012\nBasic reporting\nNo comment\nExperimental design\nNo comment\nValidity of the findings\nNo comment\nAdditional comments\nThis article addresses an important topic. Although the authors write in their conclusion that formal reporting guidelines might be premature, it might be helpful to provide a flexible sample rubric or an adaptable list of elements to include and/or report. This sample list could be designed in the spirit of the Dublin Core which enables archivists to classify items according to a flexible list of accepted search terms, which can be adapted to fit the specifics of various projects by content and context.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"A systematic review of methods for studying consumer health YouTube videos, with implications for systematic reviews (v0.1)\". PeerJ https://doi.org/10.7287/peerj.147v0.1/reviews/2",
    "pdf_3": "https://peerj.com/articles/147v0.1/submission",
    "all_reviews": "Review 1: Fred Muench \u00b7 Aug 6, 2013 \u00b7 Academic Editor\nACCEPT\nThank you for making the changes to the introduction - the paper will be useful to help understand this research area. The paper can be accepted but please make the following changes:\n1. Please copy edit the entire manuscript. There were several typos\n2. Line 24: At present little is known about.... I would start the paragraph with - line 27 - We wished to conduct a systematic review -- first. Then At present... (Switch order). That will help transition the reader - then leading to why you did this preliminary paper.\n\nCongratulations.\nReview 2: Fred Muench \u00b7 Mar 19, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nThank you for making the edits to the document. It enhances the manuscript. My main concern at this point is the introduction. It is now extremely short and the paragraphs do not necessarily flow after items were deleted without modifying the text - and new text was not included. I apologize if this was not clear in the previous edit but is important for the best possible manuscript. I believe the introduction should at minimum:\n\n1- have an additional paragraph on how health videos are being used presently across health disciplines and the information/intervention they are attempting to convey to consumers. For example, it is post-op care instructions, general health advice, etc. Please give examples.Even though this is a description of reviews this will help orient the reader who may not be experts.\n\n2- Your main question is the following: \"In reviews and systematic reviews of health-related YouTube videos, what are common reporting practices, exemplary methods and methodological challenges.\"\n\nAfter the above inserted paragraph, please write an additional paragraph on the problem or challenge you are trying to address with your paper -- what discrepancies exist, why conduct this review aside from the prevalence of these videos for example. More information on the importance of this review is also important to orient the reader.\n\nIn general the background should be clearer and set the reader up for your methods.\n\nBased on all your research, this should be a minor revision. Please contact me with questions. Fred\nReview 3: Fred Muench \u00b7 Jan 17, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nAs with the two reviewers, I believe this article has merit and should be accepted with revisions. Understanding how to evaluate consumer health videos is an important area of study which will only grow . Please be sure to copy edit the manuscript as there are several errors. I have three broad based suggestions (some corresponding to reviewers) that may improve the manuscript for the reader and several smaller ones. First, the introduction appears to focus more of the background of how you came to the decision to study health videos by focusing on infant pain which is never mentioned again. It would be helpful to discuss in more detail about who is creating these videos, more on why this is an important area of study and most importantly to bring it back to your conclusion and goals \u2013 what are the current accepted systematic review methods (lines 316) and why we need to modify these current methods for videos and create a new method. In the method section, please merge how you searched into one paragraph (e.g. add 61-63 into the paragraph). Please define \u201csnowball techniques\u201d in more detail. Line 138 appears to be missing some text. The review provided so much important information as presented in the table and was a well done descriptive study. As with reviewer two, it would be nice to see you expand the discussion and conclusions based on your knowledge and findings to include \u2013 what are the most important areas that researchers cover when conducting reviews of Web-based health videos beyond the descriptive table of what you covered, why it is important and include a sample rubric, and with your knowledge, it would be very interesting to include a brief paragraph on what individuals developing videos should take into account - though this is clearly a separate paper and just a suggestion to enhance the paper for a larger audience. Looking forward to seeing the revised manuscript.\nReview 4: Reviewer 1 \u00b7 Jan 16, 2013\nBasic reporting\nWhile I am unfamiliar with the journal, the research question seems well-identified, contained, and sized.\nExperimental design\nThe research question is clear. This study is strictly descriptive, which is fine for addressing the authors' research question. Methods were well-described.\nValidity of the findings\nIt seems likely that these findings are valid, inasmuch as the methods for the present review are well-described.\nAdditional comments\nThis paper is a useful description of \u201creviews of reviews\u201d of YouTube videos related to health. It specifies many dimensions of review reporting, and in some cases makes recommendations (e.g., for stopping rules). As information sources grow in channel types, attempts to summarize it will need to develop standards for reporting, and papers such as this one will help to make this happen.\nIt is interesting that some institutions required IRB review for the studies and some did not; were there differences between the two categories that account for this?\nOne issue that puzzles me a bit has to do with \u201csnowballing\u201d to include videos that appear as recommendations alongside the \u201cindex\u201d video. It seems that using this procedure would result in non-replicable results (if replicability is even a relevant issue here), and would interfere with systematicity in the search. A description of how this strategy could be made more systematic would be helpful.\nI would request that the authors attend carefully to their use of the terms \u201creview\u201d and \u201cstudy\u201d. Sometimes reviews are called studies, and this is confusing: for example, in line 260 where the study that is referred to is the pain-during-vaccination one that initiated this project.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"A systematic review of methods for studying consumer health YouTube videos, with implications for systematic reviews (v0.1)\". PeerJ https://doi.org/10.7287/peerj.147v0.1/reviews/1\nReview 5: Reviewer 2 \u00b7 Dec 29, 2012\nBasic reporting\nNo comment\nExperimental design\nNo comment\nValidity of the findings\nNo comment\nAdditional comments\nThis article addresses an important topic. Although the authors write in their conclusion that formal reporting guidelines might be premature, it might be helpful to provide a flexible sample rubric or an adaptable list of elements to include and/or report. This sample list could be designed in the spirit of the Dublin Core which enables archivists to classify items according to a flexible list of accepted search terms, which can be adapted to fit the specifics of various projects by content and context.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"A systematic review of methods for studying consumer health YouTube videos, with implications for systematic reviews (v0.1)\". PeerJ https://doi.org/10.7287/peerj.147v0.1/reviews/2\nReview 6: \n",
    "LLM_extracted_review": "['Review 1: The paper will be useful to help understand this research area. Please copy edit the entire manuscript. There were several typos. The introduction should start with \"We wished to conduct a systematic review\" to help transition the reader.\\n\\nReview 2: The introduction is now extremely short and lacks flow after deletions. An additional paragraph on how health videos are currently used across health disciplines is needed. The main question should be clearly stated, and a paragraph on the problem or challenge being addressed should be included to orient the reader.\\n\\nReview 3: The introduction focuses too much on infant pain, which is not mentioned again. More detail on who creates these videos and why this area is important is needed. The methods section should merge how the search was conducted into one paragraph, and \"snowball techniques\" should be defined in more detail. The discussion should expand on important areas for researchers and include suggestions for video developers.\\n\\nReview 4: The research question is well-identified and the methods are well-described. The paper is a useful description of reviews of YouTube videos related to health. There are concerns about the \"snowballing\" method used for including videos, as it may lead to non-replicable results. The use of the terms \"review\" and \"study\" should be clarified.\\n\\nReview 5: Although formal reporting guidelines might be premature, providing a flexible sample rubric or adaptable list of elements to include could be helpful. This could be designed similarly to the Dublin Core for classification.']"
}