Modification of chimeric (2S, 3S)-butanediol dehydrogenase based on structural information.
PROTEIN AND PEPTIDE LETTERS(2015)
摘要
A chimeric (2S, 3S)-butanediol dehydrogenase (cLBDH) was engineered to have the strict (S)-configuration specificity of the (2S, 3S)-BDH (BsLBDH) derived from Brevibacterium saccharolyticum as well as the enzymatic stability of the (2R, 3S)-BDH (KpMBDH) from Klebsiella pneumonia by swapping the domains of two native BDHs. However, while cLBDH possesses the stability, it lacks the specificity. In order to assist in the design a BDH having strict substrate specificity, an X-ray structural analysis of a cLBDH crystal was conducted at 1.58 angstrom. The results obtained show some readily apparent differences around the active sites of cLBDH and BsLBDH. Based on this structural information, a novel (2S, 3S)-BDH having a preferred specificity was developed by introducing a V254L mutation into cLBDH. The influence of this mutation on the stability of cLBDH was not evaluated. Nevertheless, the technique described herein is an effective method for the production of a tailor-made BDH.
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关键词
2,3-butanediol,butanediol dehydrogenase,domain chimera,short-chain dehydrogenase/reductase family,stereo-specificity,tailor-made enzyme,X-ray structural analysis
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