Esterases with an Introduced Amidase‐Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity

Syrén, Per‐Olof; Hendil-Forssell, Peter; Aumailley, Lucie; Besenmatter, Werner; Gounine, Farida; Svendsen, Allan; Martinelle, Mats; Hult, Karl

doi:10.1002/cbic.201100779

Cited by 31 publications

(37 citation statements)

References 16 publications

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“…Mechanism for the cleavage of acetone cyanohydrin by ( S )-HNL from Hevea brasiliensis 39,95 . Blocking of the oxyanion hole by threonine forces the acetone carbonyl group into a different orientation from that for the ester carbonyl group in esterases.…”

Section: Figurementioning

confidence: 99%

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

2015

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Enzymes within a family often catalyze different reactions. In some cases, this variety stems from different catalytic machinery, but in other cases the machinery is identical; nevertheless, the enzymes catalyze different reactions. In this review, we examine the subset of α/β-hydrolase fold enzymes that contain the serine-histidine-aspartate catalytic triad. In spite of having the same protein fold and the same core catalytic machinery, these enzymes catalyze seventeen different reaction mechanisms. The most common reactions are hydrolysis of C–O, C–N and C–C bonds (Enzyme Classification (EC) group 3), but other enzymes are oxidoreductases (EC group 1), acyl transferases (EC group 2), lyases (EC group 4) or isomerases (EC group 5). Hydrolysis reactions often follow the canonical esterase mechanism, but eight variations occur where either the formation or cleavage of the acyl enzyme intermediate differs. The remaining eight mechanisms are lyase-type elimination reactions, which do not have an acyl enzyme intermediate and, in four cases, do not even require the catalytic serine. This diversity of mechanisms from the same catalytic triad stems from the ability of the enzymes to bind different substrates, from the requirements for different chemical steps imposed by these new substrates and, only in about half of the cases, from additional hydrogen bond partners or additional general acids/bases in the active site. This detailed analysis shows that binding differences and non-catalytic residues create new mechanisms and are essential for understanding and designing efficient enzymes.

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Section: Figurementioning

confidence: 99%

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

2015

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“…Two different strategies to discover novel enzyme scaffolds with enhanced promiscuous activity. Left: Boosting promiscuous activity by focusing on the in silico discovery [23,24] and engineering [34] of key mechanistic structural elements in modern enzymes is represented by the forward gear. This is exemplified by focusing on a hydrogen bond acceptor (arrow) that could act as a shifter for enhanced amidase over esterase activity by facilitating nitrogen inversion (structure shown), a key step in amide bond hydrolysis [26].…”

Section: Resultsmentioning

confidence: 99%

“…Amide synthesis was achieved as previously described [34] by acid chloride chemistry using p-nitroaniline and butyryl chloride. Enzymatic hydrolysis was followed spectrophotometrically at 410 nm in phosphate buffer (50 mM KH 2 PO 4 /K 2 HPO 4 , pH 7.5) at 37˝C.…”

Section: Enzyme Activity and Kinetic Datamentioning

confidence: 99%

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

2016

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Abstract:The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the α/β phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.

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“…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) (Patkar et al, 1997), introduction of space to accommodate the substrate (W104, L278, A282, I285, V286), introduction of dipolar interactions between the enzyme and the substrate (A132, A141, I189) (Syrén et al, 2012) 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.…”

Section: Methodsmentioning

confidence: 99%

In silicoscreening of 393 mutants facilitates enzyme engineering of amidase activity in CalB

Hediger

Vico

Rannes

et al. 2013

PeerJ

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Our previously presented method for high throughput computational screening of mutant activity (Hediger et al., 2012) 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 ≥3-fold wild type activity and seven out of the eight least active mutants with ≤0.5-fold wild type activity. The method is further used to screen all sterically possible (386) double-, triple- and quadruple-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified.

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Esterases with an Introduced Amidase‐Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity

Cited by 31 publications

References 16 publications

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

In silicoscreening of 393 mutants facilitates enzyme engineering of amidase activity in CalB

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