Title Regulatory role of cysteines in (2R, 3R)-butanediol dehydrogenase BdhA of Bacillus velezensis strain GH1-13
Author Yunhee Choi1 and Yong-Hak Kim2*
Address 1Agricultural Microbiology Division, National Institute of Agricultural Science, Rural Development Administration, Wanju 55365, Republic of Korea, 2Department of Microbiology, Daegu Catholic University School of Medicine, Daegu 42472, Republic of Korea
Bibliography Journal of Microbiology, 60(4),411-418, 2022,
DOI 10.1007/s12275-022-2018-y
Key Words Plant growth-promoting rhizobacterium, Bacillus velezensis, (2R, 3R)-butanediol dehydrogenase, zinc binding motif, site-directed mutagenesis
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.