Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis

Abstract

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.

Unveiling the Catalytic Mechanism of a Processive Metalloaminopeptidase

“…Importantly, care must be taken to interpret metal occupancy in structural data, as it can be influenced by crystallization conditions and not necessarily reflect protein activation state, metal binding stoichiometry, and catalysis. 50 Pre steady-state multiple turnover (MTO) experiments were carried out in the presence of excess Leu-AMC while varying the ratio of MnCl 2 :PaPepA (Figure 4). A broad range of metal:enzyme ratios was investigated to explore the difference between K D and K ACT for PepA-mediated catalysis when using Mn 2+ (Figure 4C,D).…”

“…Crystal structures depicting two metals in the active site and the difference in magnitude for K ACT and K D values led us to hypothesize that metal occupancy in one site was not sufficient for activation. Importantly, care must be taken to interpret metal occupancy in structural data, as it can be influenced by crystallization conditions and not necessarily reflect protein activation state, metal binding stoichiometry, and catalysis …”

Inhibitors of riboflavin biosynthetic pathway enzymes as potential antibacterial drugs

“…The detailed investigation of DHBPS structures in complexes with several metals highlighted the breathing of metal or shift in the position of two metals during the course of the reaction ( Liao et al, 2002 ; Islam et al, 2015 ). Recently, two transient intermediates were identified using time-dependent structural studies and helps in defining the mechanism of DHBPS ( Kenjić et al, 2022 ).…”