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RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate for lumazine synthase that is incorporated into the xylene moiety of lumazine and ultimately the riboflavin isoalloxazine. The reaction was first identified by Bacher and co-workers in the 1990s, and their chemical mechanism hypothesis became canonical despite minimal direct evidence. X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues, and the consensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentrations equal to the enzyme concentration. This was confirmed by electron paramagnetic resonance of the enzyme reconstituted with manganese and crystal structures liganded with Mn2+ and a variety of sugar phosphates. Two transient species prior to the formation of products were identified using acid quench of single turnover reactions in combination with NMR for singly and fully 13C-labeled d-Ru5P. These data indicate that dehydration of C1 forms the first transient species, which undergoes rearrangement by a 1,2 migration, fusing C5 to C3 and generating a hydrated C4 that is poised for elimination as formate. Structures determined from time-dependent Mn2+ soaks of VcRibB-d-Ru5P crystals show accumulation in crystallo of the same intermediates. Collectively, these data reveal for the first time crucial transient chemical states in the mechanism of RibB. 

RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excis-es the C4 of D-ribulose-5-phosphate (D-Ru5P) as formate. This chemistry forms the four-carbon sub-strate for RibE (lumazine synthase) that is incorporated into the xylene moiety of lumazine and ulti-mately the riboflavin isoalloxazine. The reaction was first identified in studies by Bacher and cowork-ers in the early 1990s and a chemical mechanism hypothesis offered by these researchers has become the consensus mechanism despite minimal direct evidence. In addition, X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues and the concensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentra-tions equal to the enzyme concentration indicating that only one metal ion is required for catalysis. This was confirmed from EPR of the enzyme reconstituted with manganese and crystal structures ob-tained from soaking with the native substrate, D-Ru5P. These data definitively show the involvement of a single active site metal ion. The slow rate of turnover of VcRibB was used to identify two transient species prior to the formation of products using acid quench of single turnover reactions in combina-tion with NMR for singly and fully 13C-labelled D-Ru5P. The data indicate that dehydration of C1 forms the first transient species that then undergoes rearrangement by a 1,2 migration that fuses C5 to C3 and renders C4 hydrated as a gem diol that is poised for elimination as formate. Time-dependent Mn2+ soaks of VcRibB-D-Ru5P co-crystals provided structures that show accumulation in crystallo of the same intermediate states as observed with acid-quench and NMR. Collectively these data reveal for the first time crucial transient chemical states in the mechanism of RibB.