Bacterial RibG is a potent target for antimicrobial agents, because it catalyzes consecutive deamination and reduction steps in the riboflavin biosynthesis. In the N-terminal deaminase domain of Bacillus subtilis RibG, structure-based mutational analyses demonstrated that Glu 51 and Lys 79 are essential for the deaminase activity. In the C-terminal reductase domain, the complex structure with the substrate at 2.56-Å resolution unexpectedly showed a ribitylimino intermediate bound at the active site, and hence suggested that the ribosyl reduction occurs through a Schiff base pathway. Lys 151 seems to have evolved to ensure specific recognition of the deaminase product rather than the substrate. Glu 290 , instead of the previously proposed Asp 199 , would seem to assist in the proton transfer in the reduction reaction. A detailed comparison reveals that the reductase and the pharmaceutically important enzyme, dihydrofolate reductase involved in the riboflavin and folate biosyntheses, share strong conservation of the core structure, cofactor binding, catalytic mechanism, even the substrate binding architecture.Flavin coenzymes are essential for a wide variety of physiological processes particularly the redox reactions and hence are ubiquitously found in all organisms (1). The precursor riboflavin is biosynthesized in plants and many microorganisms (2, 3). The imidazole ring of GTP is first hydrolytically opened with elimination of formate by GTP cyclohydrolase II to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5Ј-phosphate (DAROPP).2 In eubacteria and plants, DAROPP is deaminated into 5-diamino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5Ј-phosphate (AROPP) and subsequently reduced into 5-amino-6-ribitylamino-2,4 (1H,3H)-pyrimidinedione 5Ј-phosphate (ARIPP) ( Fig. 1) (4,5). In most eubacteria, the responsible enzyme is a bifunctional protein such as Bacillus subtilis RibG (BsRibG), which is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain). In contrast, in fungi and some archaea, DAROPP is first reduced into 2, 5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5Ј-phosphate (DARIPP) and then deaminated into ARIPP. The responsible enzymes are two separate proteins, Rib7 and Rib2 (6, 7). Furthermore, animals lack this biosynthetic pathway and hence must obtain riboflavin from dietary sources. Thus the enzymes of the riboflavin biosynthesis pathway have a strong possibility of becoming novel antimicrobial targets, particularly for the development of new chemotherapeutic agents to help defend against antibiotic-resistant pathogens (8, 9).Recently, we have solved the tetrameric ring-like structure of BsRibG (10). The D domain belongs to the cytidine deaminase superfamily, in which the members catalyze the hydrolytic deamination of the base moiety of a variety of nucleotides including RNA, DNA, mononucleotides, and several therapeutically useful analogues (11). Despite no significant sequence similarity, the R domain is the only protein so far to share high structural homol...

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