N-methyl-bacillithiol, a new metabolite discovered in theChlorobiaceae, indicates that bacillithiol and derivatives are widely phylogenetically distributed

Abstract

AbstractLow-molecular weight (LMW) thiols are metabolites that mediate redox homeostasis and the detoxification of chemical stressors in cells. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including theChlorobiaceae. Fluorescent thiol labeling of metabolite extracts coupled with HPLC showed thatChlorobaculum tepidumcontained a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C14H24N2O10S. These data suggested the new thiol is closely related to bacillithiol (BSH), the major LMW thiol from low G+C% Gram-positive bacteria. By comparing the as-isolated bimane adduct with chemically synthesized candidate structures, theCba. tepidumthiol structure was identified as N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, a rarely observed modification in metabolism. Orthologs of bacillithiol biosynthetic genes in theCba. tepidumgenome were required for the biosynthesis of N-Me-BSH. Furthermore, the CT1040 gene product was genetically identified as the BSH N-methyltransferase. N-Me-BSH was found in allChlorobiexamined as well asPolaribactersp. strain MED152, a member of theBacteroidetes.A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of theChlorobi,Bacteroidetes,Deinococcus-Thermus, andFirmicutes, but also byAcidobacteria,Chlamydiae,Gemmatimonadetes, andProteobacteria.Significance StatementHere, N-Me-BSH is shown to be a redox-responsive LMW thiol cofactor inCba. tepidumand the gene,nmbA, encoding the BSH N-methyltransferase responsible for its synthesis is identified. The co-occurrence of orthologs to BSH biosynthesis genes and bacillithiol N-methyltransferase was confirmed to correctly predict LMW thiol biosynthesis in phylogenetically distant genomes. The analysis indicates that BSH/N-Me-BSH are likely the most widely distributed class of LMW thiols in biology. This finding sheds light on the evolution of LMW thiol metabolism, which is central to redox homeostasis, regulation and stress resistance in all cellular life. It also sheds light on a rare chemical modification. N-Me-BSH is the fourth instance of cysteinyl nitrogen methylation in metabolism. Identification of the BSH N-methyltransferase reported here will enable detailedin vivoandin vitrodissection of the functional consequences of this modification. As a standalone N-methyltransferase, NmbA may be useful as a component of constructed biosynthetic pathways for novel product (bio)synthesis.