Cloning, homology modeling, and reaction mechanism analysis of a novel cis-epoxysuccinate hydrolase from Klebsiella sp.

Abstract

The gene encoding a novel cis-epoxysuccinate hydrolase, which hydrolyzes cis-epoxysuccinate to L (+)-tartaric acid, was cloned from Klebsiella sp. BK-58 and expressed in Escherichia coli. The ORF was 825 bp encoding a mature protein of 274 amino acids with a molecular mass of 30.1 kDa. Multiple sequence alignment showed that the enzyme belonged to the haloacid dehalogenase-like super family. Homology modeling and site-directed mutagenesis were performed to investigate the structural characteristics of the enzyme. Its overall structure consisted of a core domain formed by six-stranded parallel β-sheets flanked by seven α-helices and a subdomain that had a four helix bundle structure. Residues D48, T52, R85, N165, K195, Y201, A219, H221, and D224 were catalytically important forming the active pocket between the two domains. An (18)O-labeling study suggested that the catalytic reaction of the enzyme proceeded through a two-step mechanism.

Single point mutations enhance activity of cis-epoxysuccinate hydrolase

“…We used classical directed evolution techniques to improve the ESH catalytic activity and the relative activity was significantly increased (230 %) by a single mutation, Phe10Gln. In addition, according to the sequence alignments for ESH (Cheng et al 2014a), we speculated that the point Phe10 may be an important catalytic site of the enzyme, which still needs specific research of protein crystal structures.…”

“…According to previous reports (Cheng et al 2014a), ESH has an a/b hydrolase fold and nine conserved catalytic residues (Carr and Ollis 2009;Ollis et al 1992;Porter et al 2015). Therefore, we started from the six conserved sites (Thr52, Arg85, Asn165, Lys195, Tyr201 and Ala219) except for the highly conserved catalytic triad Asp48-His221-Asp224.…”

Enantiomeric Tartaric Acid Production Using cis-Epoxysuccinate Hydrolase: History and Perspectives

“…BK-52 and CESH[L]s from Rhodococcus opacus , Nocardia tartaricans , and Klebsiella sp. BK-58, have also been sequenced and cloned [17,19,41,42,43]. The genes and the derived amino acid sequences of CESHs provided the basis for later studies of the recombinant expression, structure, and mechanisms as well as the protein engineering of CESHs.…”

“…Analyses of the amino acid sequences of CESHs indicated that CESH[D]s and CESH[L]s are completely different proteins [19]. Subsequent structural analysis and mechanism studies revealed that they have different structures and catalysis mechanisms [41,43,44,45]. In a recent study [46], we determined a high-resolution structure and elucidated detailed catalytic mechanisms for CESH[D], but these characteristics have not yet been reported for CESH[L].…”

“…BK-52 [17,19,37,40,43]. Some of these genes have been successfully expressed in Escherichia coli [17,19,37,43]. The amino acid sequences of CESH[L] from Rhodococcus opacus and Nocardia tartaricans CAS-52 are identical, but they only share 36% sequence identity with the CESH[L] from Klebsiella sp.…”