Biocatalysis has expanded rapidly in the last decades with the discoveries of highly stereoselective enzymes with broad substrate specificity. A new frontier for biocatalysis is broad reaction specificity, where enzymes catalyze alternate reactions. Although often under-appreciated, catalytic promiscuity has a natural role in evolution and occasionally in the biosynthesis of secondary metabolites. Examples of catalytic promiscuity with current or potential applications in synthesis are reviewed here. Combined with protein engineering, the catalytic promiscuity of enzymes may broadly extend their usefulness in organic synthesis.

Catalytic Promiscuity in Biocatalysis: Using Old Enzymes to Form New Bonds and Follow New Pathways

During the last three decades the use of hydrolases for the catalysis of environmentally friendly organic processes under mild reaction conditions has been well documented. Hydrolases have shown themselves to be ideal tools for the acceleration of synthetic transformations because of their high stability, catalytic efficiency, commercial availability and broad substrate specificity in a wide spectrum of biocatalyzed processes. In recent years, novel examples have appeared related to non-conventional reactions catalyzed by hydrolytic enzymes. Amongst these, lipases and acylases have gained much attention as promiscuous biocatalysts showing good levels of reactivity in C–C bond formation, C–heteroatom bond formation, oxidative processes, and novel hydrolytic reactions. This critical review covers recent investigation in the field of catalytic promiscuity, and highlights the most surprising and uncommon activities that this class of enzymes shows in organic synthetic transformations (111 references).

Hydrolases: Catalytically promiscuous enzymes for non-conventional reactions in organic synthesis

Biocatalysis as a profound tool in the preparation of highly enantiopure β-amino acids

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.

EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity

Hydrolases in the Stereoselective Synthesis of N-Heterocyclic Amines and Amino Acid Derivatives

The alkaline hydrolysis of (3S,5R,6R)-methyl benzylpenicilloate, and the corresponding carboxamide and N-ethylamide, is accompanied by an absorbance increase at 285 nm. This is attributed to a competing elimination reaction across C6–C5 to open the thiazolidine ring and reversibly generate an enamine intermediate. Kinetic analysis and hydrolysis in D2O do not indicate a significant build-up of this intermediate during hydrolysis of the methyl ester. However, over the pH range 4–11 the rate of thiazolidine ring opening is competitive with hydrolysis of the ester function. The deuterium solvent kinetic isotope effect on the ring closure reaction is 7.5.

Thiazolidine ring opening in penicillin derivatives. Part 2. Enamine formation

Porcine liver esterase selectively catalyses the hydrolysis of the β-lactam of the methyl ester of benzylpenicillin with a Kcat/Km of 675 dm3 mol–1 s–1 and without hydrolysing the methyl ester function.

An esterase with β-lactamase activity

α-Methyl (3S,5R,6R)benzylpenicilloate is a reversible competitive inhibitor of β-lactamase l from Bacillus cereus; the inhibition constant Ki is 6.38 × 10–4M and is independent of pH between pH 5 and 8 which is compatible with binding of the free base form of the thiazolidine residue to the enzyme.

Mark Jones

Papers

Thiazolidine ring opening in penicillin derivatives. Part 2. Enamine formation

An esterase with β-lactamase activity

A reversible inhibitor of β-lactamase I from Bacillus cereus

Thiazolidine Ring Opening in Penicillin Derivatives. Part 1. Imine Formation.