Supporting Information

Quantum mechanical (QM) calculations

The initial coordinates of the cluster model of Candida antarctica lipase B (CalB) was based on the PDB file 1LBT (CalB with Tween80). The QM calculations were performed by density functional theory (DFT) using the B3LYP hybrid density functional1 as implemented in US-GAMESS version 1 MAY 2013 (R1)2. Geometry optimizations were carried out at the B3LYP/6-31G(d,p) level of theory and by using the convergence criterion that the largest component of the gradient should be less than 5*10-4 Hartree/Bohr. Atoms corresponding to backbone positions were kept fixed during the calculations (see Figure S1). The sp2-like transition state for nitrogen inversion during the acylation of N-ethyl butyramide was constructed as previously described.3 A water molecule as potential hydrogen bond acceptor was placed within vicinity of the scissile NH-group of the substrate, and full geometry optimization was then performed (see Figure S2). Additional solvation of the active site of the wild type and I189A variant by a second water molecule was achieved by placing the additional solvent molecule towards the entrance of the active site and within hydrogen bonding distance of the first water. The water cluster and the active site model was then allowed to relax by full geometry optimization as described above.

Figure S1. Model used for the quantum mechanical calculations on CalB. Atoms labelled with an asterisk correspond to backbone positions and were kept fixed during the quantum chemical cluster simulations. Parts of the substrate (N-ethyl butyramide) are shown in enlarged sticks.

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2015

Figure S2. In silico design of a water cluster to achieve efficient TS stabilisation. Geometry optimizations were performed using DFT at the B3LYP/6-31G(d,p) level of theory. A) The TS for nitrogen inversion in CalB solvated with one water molecule (wat), that was placed in vicinity of the substrate amide group prior to minimization. The residue 189 displays a backbone carbonyl (encircled) residing in a spatial position that is antiperiplanar to the bond formed between the catalytic Ser and the former carbonyl carbon of the substrate, a geometrical arrangement of the hydrogen bond acceptor found in amidases and proteases.4 For clarity, the catalytic serine is labelled and hydrogen bonds in the oxyanion hole are shown. Parts of the substrate are shown in enlarged sticks. The introduced key hydrogen bond between the scissile NH-group and the stabilised water cluster is shown by the arrow. B) Quantum mechanical calculations on the wild-type enzyme solvated with two water molecules. Upon DFT geometry optimization, the water cluster resides in an unproductive conformation for hydrogen bond formation to the TS. Parts of the substrate (N-ethyl acetamide) is shown for clarity. C) DFT geometry optimization for the I189A variant with a deprotected backbone.

Molecular dynamics (MD) simulation

Computer modeling on CalB was based on the PDB file 1LBT using YASARA5 (Yet Another Scientific Artificial Reality Application) version 14.5.21. The PDB file was preprocessed by adding all missing hydrogens and removing the N-Acetyl-D-Glucosamine on Asn74. Subsequently the structure was minimized through repeated steps of short molecular dynamics and energy minimizations, initially on all hydrogens, then by releasing fixed waters and finally on all atoms. The variants were constructed from this refined structure and minimized again. The second tetrahedral intermediate during the acylation step of amide hydrolysis was modelled as an analogue for the transition state of nitrogen inversion. The model was constructed by covalently attaching the substrate to Ser105. Simulations were performed in a waterbox of dimensions 54 Åx65 Åx63 Å that was neutralized at pH 7.4 by the addition of 0.9% NaCl. Before the start of the simulations, all water molecules within a radius of 8 Å from the scissile NH-group of the substrate were removed. The AUTOSMILES6 methodology was used and PME7 accounted for long range electrostatic interactions. The cut-off for Van der Waals interactions was set to 7.86 Å. The simulations were run over 100 ns using Amber03 force field and snapshots were taken every 2.5 ps. All MD simulations were run in duplicate using different initial random seeds, which did not affect the conclusions. All values presented from the MD runs are the average from the two independent simulations. The snapshots were analysed in respect of distances and angles for the hydrogen bonded water networks connecting the scissile NH-group of the substrate with the enzyme.

Figure S3. Analysis of water clusters. The relative probability of hydrogen bond formation between the reacting NH-group of the substrate (in the second tetrahedral intermediate representing the TS for nitrogen inversion) and protein backbone and sidechains via water was studied by 100 ns MD. Water clusters consisting of up to four water molecules were considered. (a) Data for wild-type enzyme. (b) Data for the I189A variant with an exposed backbone (c) Data for the control I189V. NH – backbone amide, C=O backbone carbonyl.

Table S1. Average number of water molecules in the active site and average number of hydrogen bonds per water molecule based on 100 ns MD. The given values are the averages from two independently run MD-simulations.

Variant Average number of waters in active site[a] Average number of hydrogen bonds per water

wild type 10.3 2.63I189A 11.8 2.80I189G 10.0 2.51I189N 11.2 2.90I189S 12.2 2.84

ControlI189V[b] 11.7 2.68

[a] The number of waters within 8 Å of the scissile NH-group of the substrate. [b] Based on 50 ns MD.

Table S2. Probability of H-bond formation from the scissile NH-group to a water molecule and the average number of hydrogen bonds donated and accepted by this key solvent molecule. The probability is based on data from 100 ns of MD and the given values are the averages from two independently run MD-simulations.

Variant Probability of H-bond from NH to a water molecule [%]

Average number of hydrogen bonds donated/accepted for the water molecule

acceptorwild type 8.9 2.08

I189A 9.4 2.35I189G 0.7 2.25I189N 12.7 2.45I189S 12.1 2.30

ControlI189V[a] 5.2 1.82

[a] Based on 50 ns MD.

Figure S4. Relative abundance of dihedral angle populations for MD snapshots with a hydrogen bond between the scissile NH-group of the substrate and a water molecule. The threshold dihedral angle (i.e. between the former carbonyl oxygen, former carbonyl carbon and the scissile NH-group of the amide substrate) for productive nitrogen inversion transition states (> 130˚) is indicated by the dashed line.

Table S3. Probability of H-bond formation from the scissile NH-group to the carbonyl oxygen of the backbone at position 189 in the TS via a water network consisting of up to two water molecules. The probability is based on data from 100 ns of MD.

Variant Relative probability to form a short water bridge in the TS between the scissile NH

group and the backbone at 189 (NH---(H2O)n---O=C(189)) [%]

n = 1 n = 2 Sumwild type 0 0.51 0.5

I189A 0.02 3.57 3.6I189G 0.50 0.04 0.54[a]

I189N[b] 0.2 1.9 2.1I189S[c] 0 0.2 0.2ControlI189V[d] 0 0.08 0.08

[a] Note: with a high relative probability of forming a bridge consisting of one water. [b] and [c] Estimated by the manual analysis of 60 evenly distributed snapshots that contained a hydrogen bond in TS between the scissile NH-group and a water molecule. [d] Based on 50 ns MD.

Figure S5. Shift in the dihedral angle population for the I189A mutant as compared to wild-type enzyme. The data corresponds to a shift in the relative probability of productive transition state formation from 0.05% for wild type to 0.3% for the I189A variant. The data is for MD snapshots with a hydrogen bond between the scissile NH-group of the substrate and a water molecule. The distribution of dihedral angles for the I189V (control) was as for wild type and is omitted for clarity. I189G and I189N displayed a shifted population of dihedrals that was similar to that of I189A.

Supplementary note on water patterns

All the variants (wild type, I189A, I189G, I189S, I189N and I189V) were analysed in the same way to allow for direct comparison of the relative probability of the formation of a short water bridge in TS between the reacting NH-group and the protein backbone at position 189. As described in the methods section, water patterns were analysed in structures for which there was a hydrogen bond between the reacting NH-group and a water molecule and the relative probabilities were corrected accordingly. Duplicate MD trajectories (100 ns) were run, except that a shorter 50 ns MD was used for the control I189V. I189G and I189N that displayed enhanced promiscuous amidase activities (Table S4) showed a shifted dihedral angle population towards higher values in the TS that was very similar to that of I189A (Figure S5).

MD-simulations unravelled that the maximal backbone deshielding represented by the I189G variant was associated with the formation of a water bridge consisting of a single water molecule (relative probability of 0.5% for I189G vs 0.02% in the I189A variant, Table S3, Figure S6a,-S7a). In accordance with this observation, the I189G variant displayed a 6-fold higher absolute amidase activity compared to that of wild type and a shift in the amidase over esterase reaction specificity of 84-fold at 37 °C (Table S4-S5). For comparison, the overall relative probability for the formation of a water cluster of two water molecules, that would be associated with a higher entropic penalty, was 0.5% for wild type (and the formation of a cluster consisting of one water was not observed).

The additional functionalization displayed by I189S and I189N lead to highly complex and ordered TS structures. The probability of the formation of short water clusters between the TS and protein was estimated by the manual analysis of 60 evenly distributed snapshots (that contained a hydrogen bond between the scissile NH-group and a water molecule). The I189S substitution, representing both deshielding and sidechain functionalization, displayed a high probability to form a short water bridge between the TS and the backbone carbonyl at position 189 (Figure S6b). However, a large cluster of three or four water molecules bridging the introduced hydroxyl with the TS was simultaneously observed (Figure S6b). In fact, the overall probability of forming a short water bridge between the TS of inversion and the protein was estimated to be 0.2%, which was lower than that of wild type (Table S3). Experimental analysis showed that the I189S variant display a significantly reduced amidase activity (Table S4), in accordance with the energetic penalty that would be associated with freezing additional water molecules in the TS (Figure S7b). In contrast, the I189N variant displayed a higher probability of forming a short water bridge in the TS, one that was intermediate between that of wild type and I189A (Table S3, Figure S6b-S7b). The I189N variant was found to display slightly higher amidase activity compared to wild type, in accordance with the MD-simulations.

Table S4. Substrate specificities for hydrolysis of p-nitrobutyranilide at 37 °C.

Variant(kcat/KM)amide

[s-1 M-1]

This work

wild type 0.07

I189A 1.6

I189G 0.41

Previous work[a]

I189N 0.11

I189S 0.0002

I189V 0.05

I189Y 0.58

[a] Taken from Syrén et al.8

Table S5. Relative ester activity extracted from Syrén et al.8 The experiments for I189G/A were performed as previously described.8

Variant Relative (kcat/KM)ester

wild type 1

I189G 0.07

I189A 0.17

I189N 0.08

I189S 0.05

I189V 0.69

I189Y 0.66

Figure S6. Analysis of water clusters for additional variants. The relative probability of hydrogen bond formation between the reacting NH-group of the substrate in the second tetrahedral intermediate (representing the TS for nitrogen inversion) and protein backbone and sidechains via water was studied by 100 ns MD. Water clusters consisting of up to four water molecules were considered. (a) Data for I189G with maximal backbone deshielding. (b) Data for I189S to include functionalization of the side chain. (c) Data for I189N. NH – backbone amide, C=O backbone carbonyl.

Figure S7. Representative water clusters. (a) I189G with a bridge consisting of one water (b) I189N with a bridge of two waters (c) I189S with a bridge of four waters implicated a highly ordered TS structure. The key hydrogen bond is shown by the arrow.

Figure S8. A rotation-dependent mechanism is not feasible. (a) In addition to nitrogen inversion, rotation around the carbon-nitrogen bond (shown by the arrow) in the first tetrahedral intermediate formed during acylation could in principle be possible to generate a productive conformation of the lone pair of the reacting nitrogen atom. (b) Rotation up and (c) down leads to severe steric clashes.

Supplementary note on solvent isotope effects

The kcat/KM values for the wild-type enzyme and I189A variant were reduced roughly 2-4 times in D2O as compared to H2O (Table 1), with an associated expected increase in the Gibbs free energy of activation. The significantly lower activation enthalpy displayed by the I189A variant in D2O compared to that of wild type is in accordance with increased hydrogen bonding strength in heavy water.9 Remarkably, the thermodynamic cycle corresponding to ΔD2O-H2OΔmut-wild typeΔH‡ of -4.5 kcal*mol-1 is very close to the experimentally found change in enthalpy when transferring a water molecule from H2O to D2O (expected value -3.9 kcal*mol-1 9 and assuming that the effect of deuterated side chains is similar for wild type and the I189A variant). Furthermore, the 189A variant displayed an entropy of activation that was 5.3 kcal*mol-1 lower than that of the wild type in D2O at 26 °C. (i.e. T * Δmut-wild type, D2OΔS‡ =-5.3 kcal*mol-1), which is in accordance with the well-known fact of the higher entropic costs of freezing a free D2O molecule compared to H2O.9

Chemicals

CaCl2, ethylene diaminetetraacetic acid (EDTA), potassium phosphate (K2HPO4/KH2PO4), imidazole, tris(Hydroxymethyl)aminomethane (TRIS) ≥ 99% purity were purchased from Merck. 4-methylumbelliferone, acetonitrile for luminescence, agar, butyryl chloride, chloroform-D (99.8ATOM% D containing 0.05% tetramethylsilane), deuterium oxide (99.9 ATOM% D), p-nitroaniline, p-nitrophenyl butyrate and sucrose of ≥ 97% purity were obtained from Sigma-Aldrich. Sodium chloride (≥ 99%) and tryptone were purchased from Duchefa Biochemie. 1,4-dioxane (≥ 99.8%) was purchased from Labscan. MgSO4 (≥ 99.7%) was acquired from VWR. Toluene (≥ 99.99%) and yeast extract were purchased from Fisher Scientific. 4-methylumbelliferyl hexylphosphonate was purchased from EUCODIS. All chemicals were used without further purification.

Construction of variants

For site directed mutagenesis, the QuikChange protocol from Stratagene was used to introduce the I189A/G mutations. The CalB wt gene containing a C-terminal His-tag, was available in house in a Pet22b+ vector. Non-overlapping DNA primers were constructed and ordered from Thermo Fischer Scientific. The sequences for the forward and reverse primer in 5’-3’ direction are given below and the introduced nucleotide alterations are denoted with uppercase letters.

I189A forward primercggcgaccgacgagGCGgttcagcctcaggtgtcc

I189A reverse primercctgaggctgaacCGCctcgtcggtcgccgagtagagg

I189G forward primercggcgaccgacgagGGCgttcagcctcaggtgtcc

I189G reverse primercctgaggctgaacGCCctcgtcggtcgccgagtagagg

Protein expression

CalB wt, CalB I189A and CalB I189G was expressed and purified according to Syrén et al.8 with the use of 5 ml HisTrap™ HP-column instead of an 1 ml column. After purification the buffer was exchanged to 20 mM potassium phosphate, pH 7.4 using Amicon Ultra-15 Centrifugal Filter Units 10K MWCO (Millipore). No residual activity of the imidazole was detected in the flow-through tested with the hydrolysis of p-nitrophenyl butyrate.

Active site titration

Active site titration was done by fluorescence measurement by inhibiting the enzyme irreversible with 4-methylumbelliferyl hexylphosphonate as previously described.8,10 Typically, 10 µL of the inhibitor solution (500 µM in acetonitrile) was used.

Synthesis of p-nitrobutyranilide

The synthesis was performed as previously described11 with the adaptions from Syrén et al.8 The identity of p-nitrobutyranilide was confirmed by 1H-NMR on a Bruker AM-400.

Determination of specificity constants kcat/KM and thermodynamic analysis

The specificity constants for the hydrolysis of p-nitrobutyranilide and p-nitrophenyl butyrate were determined as described by Syrèn et al.8 with the difference that 10 µl of enzyme solution was added to the cuvettes. For the experiments in D2O, the pH of the buffer was set to 7.6 (corresponding to pD 8.0). In the thermodynamic analysis, kcat/KM values were determined at different temperatures ranging from 12-33 °C for ester hydrolysis and 25-45 °C for amide hydrolysis. The Eyring transition state theory equation was linearized and used to determine the activation enthalpies and entropies for amide- and ester hydrolysis (figure S9) giving coefficients of determination (r2) between 0.968-1.00. For the amide hydrolysis measurements, triplicates at three different temperatures were performed from which the averages were used in the plot.

0.0031 0.0032 0.0033 0.0034 0.0035 0.0036

-36

-34

-32

-30

-28

-26

-24

-22

-20

-18

-16

CalB wt (amide H2O)CalB wt (amide D2O)CalB I189A (amide H2O)CalB I189A (amide D2O)CalB wt (ester H2O)CalB I189A (ester H2O)

1/T [K-1]

ln (k

cat/

KM)/

(kB*

T/h)

Figure S9. Linearization of the Eyring equation with the second order rate constants kcat/KM. The r2 were between 0.968-1.00.

From figure S9 an enthalpy-entropy compensation plot was derived (figure S10). The data for amide hydrolysis forms nearly a straight line (r2 = 0.968), which suggest that the hydrolysis for both wild type and mutant proceeds through the same reaction mechanism12. As expected, the ester hydrolysis data does not fit on the line corresponding to amide hydrolysis, which would be in accordance with a reaction that proceeds with a different transition state (without nitrogen inversion).

wt, H2O

wt, D2O

I189A, H2OI189A, D2O

wt, H2OI189A, H2O

-0.015 -0.01 -0.005 0 0.005 0.010

5

10

15

20

25

Amide hydrolysisEster hydrolysis

ΔS‡ [kcal*mol-1*K-1]

ΔH‡

[kca

l*m

ol-1

]

Figure S10. Enthalpy-entropy compensation plot derived from figure S9. The data for amide hydrolysis in H2O and D2O forms a straight line (r2 = 0.968) which would be in accordance with a reaction that proceeds through the same reaction mechanism for CalB wt and CalB I189A.

Circular dichroism

The secondary structural elements for CalB wt and CalB I189A were analysed by circular dichroism in a J-810 spectrophotometer from JASCO. The concentration of protein used were 0.03 mg*ml-1 in 20 mM of potassium phosphate buffer, pH 7.4. The measurements started at 260 nm and made decrements of 0.2 nm until 190 nm (figure S11). Five repeated measurements were done for each sample to produce the data and the potassium phosphate buffer spectrum was subtracted from the signal. From the data deconvolution with CDNN13 (table S6) it was concluded that no major secondary structural rearrangements were introduced by the single-point mutation.

190 200 210 220 230 240 250 260

-100

-80

-60

-40

-20

0

CalB wt

CalB I189A

Wavelength [nm]N

orm

ailiz

ed re

spon

ce

Figure S11. Data from circular dichroism analysis.

Table S6. Secondary structural elements of CalB wt and I189A measured by circular dichroism at wavelength range between 190-260 nm. CDNN was used for deconvolution of the data.

Variant Secondary element [%]

Helix Antiparallel Parallel Beta-Turn Ran. Coil Total Sum

wild type 21.0 25.2 12.0 19.1 42.7 120.1

I189A 21.2 24.8 11.9 19.1 42.5 119.5

References1 a) C. Lee, W. Yang, R. G. Parr, Phys. Rev. B: Condens. Matter, 1988, 37, 785-789; b) A. D. Becke, Phys. Rev. A: Gen.

Phys., 1988, 38, 3098-3100; c) A. D. Becke, J. Chem. Phys., 1992, 96, 2155-2160; d) A. D. Becke, J. Chem. Phys., 1992, 97, 9173-9177; e) A. D. Becke, J. Chem. Phys., 1993, 98, 5648-5652.

2 M. W. Schmidt, K. K. Baldridge, J. A. Boatz, S. T. Elbert, M. S. Gordon, J. H. Jensen, S. Koseki, N. Matsunaga, K. A. Nguyen, S. Su, T. L. Windus, M. Dupuis, J. A. Montgomery, J. Comput. Chem., 1993, 14, 1347-1363.

3 P.-O. Syren, F. Le Joubioux, Y. Ben Henda, T. Maugard, K. Hult, M. Graber, ChemCatChem, 2013, 5, 1842-1853.4 P.-O. Syren, K. Hult, ChemCatChem, 2011, 3, 853-860.5 E. Krieger, T. Darden, S. B. Nabuurs, A. Finkelstein, G. Vriend, Proteins Struct. Funct. Bioinf., 2004, 57, 678-683.6 A. Jakalian, D. B. Jack, C. I. Bayly, J. Comput. Chem., 2002, 23, 1623-1641.7 U. Essmann, L. Perera, M. L. Berkowitz, T. Darden, H. Lee, L. G. Pedersen, J. Chem. Phys., 1995, 103, 8577-8593.8 P.-O. Syrén, P. Hendil-Forssell, L. Aumailley, W Besenmatter, F Gounine, A. Svendsen, M Martinelle, K. Hult,

ChemBioChem, 2012, 13, 645–648.9 C.G. Swain, R.F.W. Bader, Tetrahedron, 1960, 10, 182-199.10 A. O. Magnusson, J. C. Rotticci-Mulder, A. Santagostino, K. Hult, ChemBioChem, 2005, 6, 1051-1056.11 R. Kourist, S. Bartsch, L. Fransson, K. Hult, U. T. Bornscheuer, ChemBioChem, 2008, 9, 67-69.12 L. Liu, Q.-X. Guo, Chem. Rev., 2001, 101, 673-695.13 G. Böhm, R. Muhr, R. Jaenicke, Protein Eng., 1992, 5, 191-195.