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Journal Article
- Regulatory role of cysteines in (2R, 3R)-butanediol dehydrogenase BdhA of Bacillus velezensis strain GH1-13
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Yunhee Choi , Yong-Hak Kim
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J. Microbiol. 2022;60(4):411-418. Published online March 14, 2022
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DOI: https://doi.org/10.1007/s12275-022-2018-y
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Abstract
- Bacillus velezensis strain GH1-13 contains a (2R,3R)-butanediol
dehydrogenase (R-BDH) BdhA which converts acetoin
to R-BD reversibly, however, little is known about its regulatory
cysteine and biological significance. We performed sitedirected
mutation of three cysteines in BdhA. The C37S mutant
had no enzyme activity and the C34S and C177S mutants
differed from each other and wild type (WT). After zinc affinity
chromatography, 1 mM ZnCl2 treatment resulted in a
3-fold enhancement of the WT activity, but reduced activity
of the C34S mutant by more than 2 folds compared to the untreated
ones. However, ZnCl2 treatment did not affect the activity
of the C177S mutant. Most of the double and triple mutant
proteins (C34S/C37S, C34S/C177S, C37S/C177S, and
C34S/C37S/C177S) were aggregated in zinc resins, likely due
to the decreased protein stability. All of the purified WT and
single mutant proteins increased multiple intermolecular disulfide
bonds in the presence of H2O2 as the buffer pH decreased
from 7.5 to 5.5, whereas an intramolecular disulfide
bond of cysteine 177 and another cysteine in the CGIC motif
region was likely formed at pH higher than pKa of 7.5. When
pH varied, WT and its C34S or C177S mutants reduced acetoin
to R-BD at the optimum pH 5.5 and oxidized R-BD to
acetoin at the optimum pH 10. This study demonstrated that
cysteine residues in BdhA play a regulatory role for the production
of acetoin and R-BD depending on pH as well as
metal binding and oxidative stress.
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