Systematic screening for catalytic promiscuity in 4-oxalocrotonate tautomerase: enamine formation and aldolase activity

The enzyme 4-oxalocrotonate tautomerase (4-OT) is part of a catabolic pathway for aromatic hydrocarbons in Pseudomonas putida mt-2, where it catalyzes the conversion of 2-hydroxy-2,4-hexadienedioate(1) to 2-oxo-3-hexenedioate(2). 4-OT is a member of the tautomerase superfamily, a group of homologous proteins that are characterized by a β-α-β structural fold and a catalytic amino-terminal proline. In the mechanism of 4-OT, Pro1 is a general base that abstracts the 2-hydroxyl proton of 1 for delivery to the C-5 position to yield 2. Here, 4-OT was explored for nucleophilic catalysis based on the mechanistic reasoning that its Pro1 residue has the correct protonation state (pK(a) ～6.4) to be able to act as a nucleophile at pH 7.3. By using inhibition studies and mass spectrometry experiments it was first demonstrated that 4-OT can use Pro1 as a nucleophile to form an imine/enamine with various aldehyde and ketone compounds. The chemical potential of the smallest enamine (generated from acetaldehyde) was then explored for further reactions by using a small set of selected electrophiles. This systematic screening approach led to the discovery of a new promiscuous activity in wild-type 4-OT: the enzyme catalyzes the aldol condensation of acetaldehyde with benzaldehyde to form cinnamaldehyde. This low-level aldolase activity can be improved 16-fold with a single point mutation (L8R) in 4-OT's active site. The proposed mechanism of the reaction mimicks that used by natural class-I aldolases and designed catalytic aldolase antibodies. An important difference, however, is that these natural and designed aldolases use the primary amine of a lysine residue to form enamines with carbonyl substrates, whereas 4-OT uses the secondary amine of an active-site proline as the nucleophile catalyst. Further systematic screening of 4-OT and related proline-based biocatalysts might prove to be a useful approach to discover new promiscuous carbonyl transformation activities that could be exploited to develop new biocatalysts for carbon-carbon bond formation.     

Direct epoxidation in Candida antarctica lipase B studied by experiment and theory

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated by density functional theory calculations, and it was demonstrated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.     

An Unexpected Promiscuous Activity of 4-Oxalocrotonate Tautomerase: The cis-trans Isomerisation of Nitrostyrene

Serendipitous switch: While exploring cis-nitrostyrene as a potential electrophile in Michael-type addition reactions catalysed by the enzyme 4-oxalocrotonate tautomerase (4-OT), it was unexpectedly found that 4-OT catalyses the isomerisation of cis-nitrostyrene to trans-nitrostyrene (k(cat) /K(m) = 1.9×10(3) M(-1) s(-1) ).     

A water molecule in the stereospecificity pocket of Candida antarctica lipase B enhances enantioselectivity towards pentan-2-ol

The effect of water activity on enzyme-catalyzed enantioselective transesterification was studied by using a solid/gas reactor. The experimental results were compared with predictions from molecular modelling. The system studied was the esterification of pentan-2-ol with methylpropanoate as acyl donor and lipase B from Candida antarctica as catalyst. The data showed a pronounced water-activity effect on both reaction rate and enantioselectivity. The enantioselectivity increased from 100, at water activity close to zero, to a maximum of 320, at a water activity of 0.2. Molecular modelling revealed how a water molecule could bind in the active site and obstruct the binding of the slowly reacting enantiomer. Measurements of enantioselectivity at different water-activity values and temperatures showed that the water molecule had a high affinity for the stereospecificity pocket of the active site with a binding energy of 9 kJ mol-1, and that it lost all its degrees of rotation, corresponding to an entropic energy of 37 J mol-1 K-1.     

Modification of Substrate Specificity Resulting in an Epoxide Hydrolase with Shifted Enantiopreference for (2,3‐Epoxypropyl)benzene

Random mutagenesis targeted at hotspots of noncatalytic active‐site residues of potato epoxide hydrolase StEH1 combined with an enzyme‐activity screen allowed the isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3‐epoxypropyl)benzene. The wild‐type enzyme favored the S enantiomer with a ratio of 2.5:1, whereas the variant displaying the most radical functional changes showed a 15:1 preference for the R enantiomer. This mutant had accumulated four substitutions distributed over two out of four mutated hotspots: W106L, L109Y, V141K, and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S enantiomer combined with retained activity with the R enantiomer. The results demonstrate the feasibility of molding the stereoselectivity of this biocatalytically relevant enzyme.     

Enhancement of the promiscuous aldolase and dehydration activities of 4-oxalocrotonate tautomerase by protein engineering

Double play: The enzyme 4-oxalocrotonate tautomerase (4-OT) catalyzes not only the initial cross-coupling of acetaldehyde and benzaldehyde to yield 3-hydroxy-3-phenylpropanal, but also the subsequent dehydration of this aldol compound to yield cinnamaldehyde as the final product. Mechanism-inspired engineering provided an active site mutant (F50A) with strongly enhanced aldol condensation activity.     

国内洗涤剂用酶的研究

综述了国内洗涤剂用酶的研究、开发及应用情况，介绍了我国加酶洗涤剂生产概况及发展前景     

Enzymatic Thioxyloside Synthesis: Characterization of Thioglycoligase Variants Identified from A Site‐Saturation Mutagenesis Library of Bacillus Circulans Xylanase

Thioglycoligases are engineered enzymes for the synthesis of thioglycosides that are derived from retaining glycosidases by replacing the acid/base catalyst. The optimal choice of substitution for the acid/base mutant is currently unknown, so to investigate this question a complete acid/base library of the model glycosidase Bacillus circulans xylanase (Bcx) was generated by using site‐saturation mutagenesis. A novel screening approach combining active site titration with semiquantitative product analysis by thin layer chromatography was established and used to evaluate specific activities of each mutant enzyme within crude cell lysates. The six most active Bcx variants were analyzed in more detail, a pH optimum of 8.5 was established and the identity of reaction products was confirmed. Optimal choices for substitution were small, preferably polar amino acids such as threonine, cysteine, and serine. We discuss the resultant data in the context of previously published studies on thioglycoligases.     

Exploring the active-site of a rationally redesigned lipase for catalysis of Michael-type additions

Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant lacks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The kca values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.     

Mixed Carbonates as Useful Substrates for a Fluorogenic Assay for Lipases and Esterases

Mixed carbonates of 7‐hydroxy‐4‐methylcoumarin were shown to be a new class of probe for fluorogenic assays. They are promising substrates for fingerprinting enzyme hydrolytic activity, and proved particularly useful because of the low level of nonspecific degradation and ease of synthesis. They are highly relevant for screening lipase and esterase libraries. These advantages make umbelliferyl carbonates highly suitable substrates for high‐throughput screening. Moreover, we report the use of chiral fluorogenic carbonates as enantiopreference probes.     

离子液体中酶催化反应的研究进展

讨论了近年来离子液体作为酶催化反应介质的应用发展。说明了不同类别的酶在离子液体或水合离子液体两相体系中具有催化活性，尤其是脂肪酶，在无水的离子液体介质反应体系中不仅能保持很好的活性，其手性选择性和操作稳定性往往优于传统介质中的表现。指出了离子液体的环境相容性使其成为绿色生物反应工程新的应用溶剂。对不适于在传统溶剂体系中进行的生物催化再生和产物回收研究，离子液体溶剂已经逐渐表现出其优势。