Zhipeng Xie

Site-directed mutagenesis of epoxide hydrolase to probe catalytic amino acid residues and reaction mechanism.

Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis‐epoxysuccinate to L(+)‐tartrate. It shows low but significant similarity to haloacid dehalogenase and haloacetate dehalogenase (16–23% identity). To identify catalytically important residues, we mutated 29 highly conserved charged and polar amino acid residues (except for one alanine). The replacement of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the enzyme activity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two‐step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction mechanism.

Analysis of essential amino acid residues for catalytic activity of cis -epoxysuccinate hydrolase from Bordetella sp. BK-52

Abstractcis-Epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52, an epoxide hydrolase (EH), catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to d(−)-tartrate. The enzyme, which shows no homology to other reported EHs, belongs to the DUF849 superfamily of prokaryotic proteins, which have unknown function. Metal composition analysis revealed that the CESH is a Zn2+-dependent enzyme with an approximately 1:1 molar ratio of zinc to enzyme. The results of an 18O-labeling study suggest that the enzyme catalyzes epoxide hydrolysis by means of a one-step mechanism. We evaluated the relationship between the structure and function of the enzyme by means of sequence alignment, modeling, substrate binding, and reaction kinetics studies. The CESH has a canonical (β/α)8 TIM barrel fold, and we used site-directed mutagenesis to identify eight residues (H47, H49, R51, T82, Y138, N140, W164, and D251) as being localized to the active site or highly conserved. On the basis of these results and theoretical considerations, we identified H47 and H49 as zinc-binding ligands, and we propose that a zinc atom and R51, T82, Y138, N140, W164, and D251 are the catalytic residues and participate in substrate binding. In summary, the structure and catalytic mechanism of the CESH from Bordetella sp. BK-52 differ from those of classic EHs, which have an α/β hydrolase fold, act via a two-step catalytic mechanism, and do not require cofactors, prosthetic groups, or metal ions.

Isolation of the stable strain Labrys sp. BK-8 for L(+)-tartaric acid production.

A novel cis-epoxysuccinate hydrolase (CESH) producing strain of Labrys sp. BK-8 for production of L(+)-tartaric acid was isolated and identified. After optimization, a maximum activity of 3597 ± 151 U/g was achieved in batch culture in a 10 L fermentor. When Labrys sp. BK-8 was immobilized on κ-carrageenan, the immobilized cells showed a high conversion rate (>99%), enantioselectivity (EE > 99.5%) and storage stability (>90 d). A conversion rate of 97% was still achieved after 10 repeat batches. The CESH was stable over a broad range of temperatures (up to 45°C) and pH values (4.0-10.0). The Labrys sp. BK-8 isolate provides a new alternative with good stability for the industrial biosynthesis of L(+)-tartaric acid.