Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.
There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. 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. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis.
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.
Stephan
S
Grosse
Hélène
H
Bergeron
Akihiro
A
Imura
Jason
J
Boyd
Shaozhao
S
Wang
Kazuo
K
Kubota
Akihiko
A
Miyadera
Traian
T
Sulea
Peter C K
PC
Lau
D019943:Amino Acid Substitution / Q000235:genetics; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D055550:Protein Stability; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013696:Temperature; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics; D044366:Transition Temperature
D004269:DNA, Bacterial; D004952:Esters; D004950:Esterases
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