Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.
Bottom Line: The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3-5 °C increase in the apparent melting temperature (T(m)) of the mutants over the native BcEST that has a T(m) of 50 °C was outperformed by TtEST, a naturally occurring homologue with a T(m) of 65 °C.Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations.This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily.
Affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.Show MeSH
Mentions: In the Protein Data Bank (Berman et al., 2000), the structural homologues of BcEST include: an aryl esterase from Pseudomonas fluorescens (PDB 1VA4, 27% identity; Cheeseman et al., 2004); a C–C bond hydrolase (MphC) of the phenylpropionate degradation pathway of E. coli (PDB 1U2E, 25% identity; Dunn et al., 2005); and, a bromoperoxidase A1 from Streptomyces aureofaciens (PDB 1A8Q, 26% identity; Hofmann et al., 1998). The sequence alignment of BcEST, together with TtEST and the three structurally similar enzymes, is given in Fig. 2.
Affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.