Biochemical Characterization and Structural Basis of Reactivity and Regioselectivity Differences between Burkholderia thailandensis and Burkholderia glumae 1,6-Didesmethyltoxoflavin N-Methyltransferase

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 Bg ToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, Bth II1283 in B. thailandensis E264, is a predicted S -adenosylmethionine (SAM)-dependent N -methyltransferase that shows a low level of sequence identity to Bg ToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike Bg ToxA, Bth II1283 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 Bth II1283 with bound S -adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. Bth II1283 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 Bg ToxA. In Bth II1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in Bg ToxA, it is first directed toward an sp 2 orbital of N6 and then toward an sp 2 orbital of N1 after planar rotation of the azapteridine ring system. Furthermore, in Bth II1283, N1 is hydrogen bonded to a histidine residue whereas Bg ToxA does not supply an obvious basic residue for either N6 or N1 methylation.