Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

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Medium- and short-chain dehydrogenase/reductase gene and protein families: The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes

Short-chain dehydrogenases/reductases (SDR): the 2002 update.

We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process ∼4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.

Improving catalytic function by ProSAR-driven enzyme evolution

High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe.

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Expanding the Enzyme Universe: Accessing Non‐Natural Reactions by Mechanism‐Guided Directed Evolution

Publisher Summary This chapter discusses dehalogenase enzymes and its types. The chapter emphasizes on the microbial origin and distribution of these enzymes, their biochemical properties, and their engineering and use in biocatalysis. Dehalogenases comprise a diverse group of enzymes belonging to different phylogenetic and mechanistic classes. They have biotechnological potential because of their high-catalytic power in the cleavage of carbon–halogen bonds of toxic environmental pollutants. Dehalogenases appear with very different structural folds, reaction types, and catalytic mechanisms. For example, several reductive dehalogenases that replace a chlorine substituent by a hydrogen possess a corrinoid cofactor typical for an electron transfer mechanism, whereas haloalkane dehalogenases belong to the α/β-hydrolase fold family, a group of proteins of which most members catalyze hydrolytic reactions via a covalent alkyl-enzyme intermediate, reminiscent to the acyl-enzyme intermediate of classical serine proteases. More recently, dehalogenases have been further explored for enantioselective conversion of other halocarboxylic acids and their esters and haloalkanes, as well as for their applicability in recycling and detoxifying trichloropropane.

Biocatalysis by dehalogenating enzymes

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The kcat and Km values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s−1 and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pKa of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.

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Halohydrin Dehalogenases Are Structurally and Mechanistically Related to Short-Chain Dehydrogenases/Reductases

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 is a homotetrameric protein containing four tryptophan residues per subunit. The fluorescence properties of the enzyme are strongly influenced by halide binding. To examine the role of the tryptophans (W139, W192, W238, and W249) in halide binding and catalysis, they were individually mutated to a phenylalanine. All mutations, except for W238F, influenced the enzymatic properties. Mutating W192 to phenylalanine inactivated the enzyme and led to dissociation into dimers and monomers. In the structure of HheC, residue W139 and residue W249 from the opposite subunit are close to the active site of the enzyme. Substitution of W139 mainly affected Km values with all tested substrates and reduced the enantiopreference for p-nitro-2-bromo-1-phenylethanol. Replacing W249 increased both kcat and Km values with all tested substrates except for the (S)-enantiomer of p-nitro-2-bromo-1-phenylethanol, for which kcat was 3-fold decreased, resulting in a 6...

Steady-State Kinetics and Tryptophan Fluorescence Properties of Halohydrin Dehalogenase from Agrobacterium radiobacter. Roles of W139 and W249 in the Active Site and Halide-Induced Conformational Change†

The α-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing β-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified α-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the α-amino acid ester hydrolase is a β-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The α-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of β-lactam antibiotic acylases.

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Annet E. J. van Merode

Papers

Halohydrin Dehalogenases Are Structurally and Mechanistically Related to Short-Chain Dehydrogenases/Reductases

Steady-State Kinetics and Tryptophan Fluorescence Properties of Halohydrin Dehalogenase from Agrobacterium radiobacter. Roles of W139 and W249 in the Active Site and Halide-Induced Conformational Change†

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an α-amino acid ester hydrolase from Acetobacter turbidans