ABSTRACT
One often-cited weakness of biocatalysis is the lack of mirror-image enzymes for the formation of either enantiomer of a product in asymmetric synthesis. Enantiocomplementary enzymes exist as the solution to this problem in nature. These enzyme pairs, which catalyze the same reaction but favor opposite enantiomers, are not mirror-image molecules; however, they contain active sites that are functionally mirror images of one another. To create mirror-image active sites, nature can change the location of the binding site and/or the location of key catalytic groups. In this Minireview, X-ray crystal structures of enantiocomplementary enzymes are surveyed and classified into four groups according to how the mirror-image active sites are formed.
Subject(s)
Enzymes/metabolism , Binding Sites , Catalytic Domain , Enzymes/chemistry , Enzymes/classification , Protein Folding , Stereoisomerism , Substrate SpecificityABSTRACT
Esterase EstB from Burkholderia gladioli, which belongs to a family of esterases related to beta-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10(6) mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10(5) clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (T(m) was raised by 13 degrees C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.
Subject(s)
Burkholderia gladioli/enzymology , Directed Molecular Evolution/methods , Esterases/metabolism , Mutant Proteins/chemistry , Amino Acid Sequence , Calorimetry, Differential Scanning , Enzyme Stability , Half-Life , Kinetics , Models, Molecular , Mutagenesis , Mutant Proteins/isolation & purification , Mutation/genetics , Protein Structure, Secondary , Sequence Analysis, Protein , Transition TemperatureABSTRACT
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.