Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols

Uppenberg, J.; Öhrner, Niklas; Norin, Martin; Hult, Karl; Kleywegt, Gerard J.; Patkar, Shamkant; Waagen,; Anthonsen, T.; Jones, T. Alwyn

doi:10.1021/bi00051a035

Cited by 483 publications

(361 citation statements)

References 26 publications

(42 reference statements)

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“…T40-O γ H participates only sparsely. The available crystal structures of CAL-B [51,39] reveal that T40 accepts H-bonds from the amide side chains of Q106 and Q157 (Fig. 1), and at least one is maintained in the simulations, accompanied by a slight distortion of the backbone, also observed in the uncomplexed variants (ESM Fig.…”

Section: Simulations Of Substratesmentioning

confidence: 84%

“…This supports the previous observation that only two donors are important in the oxyanion hole at any one time, even in enzymes having donors in the sidechains. [20,21] However, as seen in the 1LBS crystal structure, [39] the covalent intermediate (and possibly the TS) binds to all three H-bond donors, perhaps by some geometric distortion induced by the tetrahedral structure.…”

Section: Simulations Of Substratesmentioning

confidence: 93%

“…I189 and I285 constitute the 'channel' dividing the active site in the small 'stereoselectivity pocket' and larger hydrophobic (or acyl) pocket. [39] This channel is the most sterically crowded region near the oxyanion hole, yet the region where the sandwichlike DA TS must be located in order to exploit the catalytic machinery of CALB. Consequently, we also modeled the I285A and S105A/I285A mutants.…”

Section: Fig 1: (A)mentioning

confidence: 99%

“…[39] The entry was chosen due to the presence of a large lipid analog covalently bound to the catalytic S105, which putatively induces a fully active conformation of the enzyme. The all-atom RMSD to two other common structures in modeling is small; 1TCA [51] = 0.29, 1LBS [39] = 0.44. The 1LBT structure was stripped of all water molecules and protonated (default protonation state) in the viewer Hermes, a part of the GOLD Suite.…”

Section: Enzyme Variantsmentioning

confidence: 99%

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Computational design of a lipase for catalysis of the Diels-Alder reaction

et al. 2010

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Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme {\em Candida Antarctica} lipase B (CALB) have been rationally engineered {\em in silico} based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like near-attack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the $\alpha$,$\beta$-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates ($v_{cat}/v_{uncat}$) of up to $10^3$ are predicted.Response to Reviewers: We are happy with the favorable comments by the reviewers. We have have corrected the manuscript as suggested by reviewer 1 1. A sentence explaining catalytic promiscuity has been added. 2. A paragraph discussing the relationship between the NAC concept and the Reaction Force has been added.We have shortened the manuscript as suggested by reviewer 2. In particular, we have moved information regarding the structural relaxation of protein structure in MD simulations to the supplementary material. Abstract Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the DielsAlder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity s...

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Section: Simulations Of Substratesmentioning

confidence: 84%

Section: Simulations Of Substratesmentioning

confidence: 93%

Section: Fig 1: (A)mentioning

confidence: 99%

Section: Enzyme Variantsmentioning

confidence: 99%

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Computational design of a lipase for catalysis of the Diels-Alder reaction

et al. 2010

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“…Starting with the X-ray crystal structure of CAL-B (1LBS), 18 we modelled the analogues of the second tetrahedral intermediate in the acetylation of the cycloalkanols, since this transition state determines the rate and selectivity in a kinetic resolution of racemic alcohols (Figure 2). …”

Section: Molecular Modellingmentioning

confidence: 99%

Is the ring conformation the most critical parameter in lipase-catalysed acylation of cycloalkanols?

2004

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CAL-B catalysed the resolution of several five and six-membered cyclic -hydroxy esters efficiently with the exception of the cis-cyclohexanol ()-4. When employing molecular modelling techniques the conformation turned out to be the most important determinant for their reactivity towards O-acetylation. In all cases, the R enantiomers reacted faster than the S enantiomers since the reactive intermediates of the former can adopt more favourable ring conformations and thus experience less steric hindrance in the active site. Furthermore, the minimised structure for the main conformer of R-4 showed that the axial hydrogens in the 3 and 5-positions with respect to the hydroxyl group prevent the enzymatic reaction.

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Alcohol inhibition and specificity studies of lipase B fromCandida antarctica in organic solvents

Garcia-Alles

Gotor

1998

Biotechnol. Bioeng.

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Alcohol inhibition of the lipase B from Candida antarctica has been studied through two different approaches: using the same inhibitor (1‐butanol) in different organic solvents and using different inhibitors (differing in chain length) in the same solvent. The competitive inhibition constant values obtained in each case correlate with the calculated activity coefficients of the substrate, suggesting that desolvation of the alcohol is the major force changed. Data dispersion observed using the second approach has been interpreted to come from contributions of enzyme–inhibitor interactions to the binding energy. On the other hand, deacylation has been found to be much less influenced by the solvent variation than the acylation step, despite of the fact that solvation of the substrate involved in this step (the alcohol) is expected to change more than for the ester. Concerning the specificity behavior of the enzyme, a bimodal pattern was observed for the deacylation rate dependence on the alcohol chain length, with the highest values for hexanol (C6) and decanol (C10). With regard to the ester specificity, ethyl caproate (C6) is the preferred one. These results have been confronted with those reported for the lipase from Candida rugosa. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 163–170, 1998.

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Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols

Cited by 483 publications

References 26 publications

Computational design of a lipase for catalysis of the Diels-Alder reaction

Computational design of a lipase for catalysis of the Diels-Alder reaction

Is the ring conformation the most critical parameter in lipase-catalysed acylation of cycloalkanols?

Alcohol inhibition and specificity studies of lipase B fromCandida antarctica in organic solvents

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