FIGURE 1. BluB catalyses DMB production.

a, Overall scheme for DMB biosynthesis and B₁₂ formation. The atoms converted to DMB are shown in red; the reaction catalysed by BluB is in the red box. b, HPLC traces (280 nm) of reactions containing 10 M BluB, 20 mM NADH and 100 M FMN, or lacking the indicated components. Similar conversion was observed in reactions containing NADPH instead of NADH (not shown). In the absence of NAD(P)H, trace amounts of product formed, probably through adventitious photoreduction of FMN followed by turnover in the active site. c, Calcofluor fluorescence of the bluB mutant. d, Growth of wild-type S. meliloti and the bluB mutant. e, Radio-TLC of the BluB reaction containing [³²P]FMN. Arrows represent migration of standards. The same result was obtained using two additional eluant systems (data not shown).