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Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.

Grosse S, Bergeron H, Imura A, Boyd J, Wang S, Kubota K, Miyadera A, Sulea T, Lau PC - Microb Biotechnol (2009)

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.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.

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Multiple sequence alignment of BcEST and TtEST with their closest structural homologues (identified by PBD codes). Identical residues are highlighted black and conserved residues on grey backgrounds. Active‐site catalytic triad residues are marked with a triangle, and mutated residues with a dot. Secondary structure elements are indicated by arrows for β‐strands and by cylinders for α‐helices, as predicted for BcEST and TtEST (shown in light‐grey above the alignment) and observed in the closest structural homologue 1VA4 (shown in dark‐grey below the alignment). For the structural homologues, indicated residue numbers are taken from the respective PDB entries.
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f2: Multiple sequence alignment of BcEST and TtEST with their closest structural homologues (identified by PBD codes). Identical residues are highlighted black and conserved residues on grey backgrounds. Active‐site catalytic triad residues are marked with a triangle, and mutated residues with a dot. Secondary structure elements are indicated by arrows for β‐strands and by cylinders for α‐helices, as predicted for BcEST and TtEST (shown in light‐grey above the alignment) and observed in the closest structural homologue 1VA4 (shown in dark‐grey below the alignment). For the structural homologues, indicated residue numbers are taken from the respective PDB entries.

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.


Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.

Grosse S, Bergeron H, Imura A, Boyd J, Wang S, Kubota K, Miyadera A, Sulea T, Lau PC - Microb Biotechnol (2009)

Multiple sequence alignment of BcEST and TtEST with their closest structural homologues (identified by PBD codes). Identical residues are highlighted black and conserved residues on grey backgrounds. Active‐site catalytic triad residues are marked with a triangle, and mutated residues with a dot. Secondary structure elements are indicated by arrows for β‐strands and by cylinders for α‐helices, as predicted for BcEST and TtEST (shown in light‐grey above the alignment) and observed in the closest structural homologue 1VA4 (shown in dark‐grey below the alignment). For the structural homologues, indicated residue numbers are taken from the respective PDB entries.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815948&req=5

f2: Multiple sequence alignment of BcEST and TtEST with their closest structural homologues (identified by PBD codes). Identical residues are highlighted black and conserved residues on grey backgrounds. Active‐site catalytic triad residues are marked with a triangle, and mutated residues with a dot. Secondary structure elements are indicated by arrows for β‐strands and by cylinders for α‐helices, as predicted for BcEST and TtEST (shown in light‐grey above the alignment) and observed in the closest structural homologue 1VA4 (shown in dark‐grey below the alignment). For the structural homologues, indicated residue numbers are taken from the respective PDB entries.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.

Show MeSH