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Identification of six important amino acid residues of MenA from Bacillus subtilis natto for enzyme activity and formation of menaquinone
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2020-08-01 , DOI: 10.1016/j.enzmictec.2020.109583 Liu-Xiu Hu 1 , Jing-Jing Feng 2 , Jing Wu 2 , Wei Li 2 , Sokhna Mbacke Gningue 2 , Zi-Ming Yang 2 , Zhou Wang 2 , Yan Liu 2 , Zheng-Lian Xue 2
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2020-08-01 , DOI: 10.1016/j.enzmictec.2020.109583 Liu-Xiu Hu 1 , Jing-Jing Feng 2 , Jing Wu 2 , Wei Li 2 , Sokhna Mbacke Gningue 2 , Zi-Ming Yang 2 , Zhou Wang 2 , Yan Liu 2 , Zheng-Lian Xue 2
Affiliation
The enzyme 1, 4-dihydroxy-2-naphthoic acid (DHNA) prenyltransferase (MenA) is a critical player in determining the efficiency of the menaquinone (MK) synthesis pathway and is an attractive target for the development of novel chemotherapeutics against pathogenic Gram-positive bacteria. However, there has been no report on structural properties or active region of MenA. To solve this challenge, we predicted the three-dimensiona structure and critical amino acid sites of MenA by bioinformatics analysis. Six amino acid sites were chosen by alligning the amino acid sequence of MenA from Bacillus subtilis natto with 4-hydroxybenzoate octaprenyl transferase (UbiA) from Escherichia coli, Aeropyrum pernix and Archaeoglobus fulgidus. Among them, four Asp sites located in two Asp-rich motifs (D78XXXXXD84 and D208XXXD212) were found to be indispensable amino acid residues in maintaining MenA activity. Site-directed mutagenesis of two other sites (Q67th, N74th) positively affected the catalytic activity of MenA and the MK titer. Q67R resulted in more than a 5-fold increase in specific 2-demethylmenaquinone (DMK) content (YP1/x) compared to wild-type, and the hydrophobic interaction between Cys63 and Arg67 could be the main reason according to the three-dimensional structure analysis. Moreover, a dramatic increase in specific MK content (YP2/x) was realized by co-expressing menG in EcMenA (Q67R). The results obtained could be useful not only in developing novel chemotherapeutics to combat potentially pathogenic Gram-positive bacteria, but also in regulating and optimizating E. coli mutant cultures for the efficient production of MK metabolites.
更新日期：2020-08-01
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