TitleBiochemical Characterization and Structural Basis of Reactivity and Regioselectivity Differences between Burkholderia thailandensis and Burkholderia glumae 1,6-Didesmethyltoxoflavin N-Methyltransferase.
Publication TypeJournal Article
Year of Publication2017
AuthorsFenwick MK, Almabruk KH, Ealick SE, Begley TP, Philmus B
JournalBiochemistry
Volume56
Issue30
Pagination3934-3944
Date Published2017 08 01
ISSN1520-4995
KeywordsBacterial Proteins, Binding Sites, Burkholderia, Catalytic Domain, Crystallography, X-Ray, Histidine, Hydrogen Bonding, Methylation, Methyltransferases, Models, Molecular, Multigene Family, Oxidation-Reduction, Phylogeny, Protein Conformation, Pyrimidinones, Recombinant Proteins, S-Adenosylhomocysteine, S-Adenosylmethionine, Species Specificity, Stereoisomerism, Triazines
Abstract

Burkholderia glumae converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to BgToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B. thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase that shows a low level of sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a class I methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine ring system is orthogonal to its orientation in BgToxA. In BthII1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in BgToxA, it is first directed toward an sporbital of N6 and then toward an sporbital of N1 after planar rotation of the azapteridine ring system. Furthermore, in BthII1283, N1 is hydrogen bonded to a histidine residue whereas BgToxA does not supply an obvious basic residue for either N6 or N1 methylation.

DOI10.1021/acs.biochem.7b00476
Alternate JournalBiochemistry
PubMed ID28665591
Grant ListS10 OD018518 / OD / NIH HHS / United States
P41 GM103485 / GM / NIGMS NIH HHS / United States