3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase (RibB) of riboflavin biosynthesis has a mononuclear magnesium active site

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Riboflavin (vitamin B2) is a precursor for the synthesis of the cofactors FAD and FMN. Like other vitamins, riboflavin cannot be synthesized by mammals and must be obtained from the diet. Bacteria and plants synthesize riboflavin in five steps using the enzymes RibA‐E. The focus of this study is the 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase, otherwise known as RibB. RibB is a magnesium dependent enzyme that catalyzes the conversion of the sugar ribulose‐5‐phosphate (Ru5P), a product of the pentose phosphate pathway, into 3,4‐dihydroxy‐2‐butanone‐4‐phosphate (DHBP). The reaction catalyzed by RibB is an unusual deformylation reaction in which the fourth carbon of the five carbon sugar is removed as formate. Bacher and colleagues1 have proposed that the mechanism is dependent on a skeletal rearrangement linking C3 and C5. We propose a fragmentation mechanism, in which the bond between carbons C3 and C4 is broken allowing formation of the new link between carbons C3 and C5 followed by release of formate. To test these competing hypotheses, we are using NMR and X‐ray crystallography to observe the intermediates formed during catalysis. We have produced 13C labeled Ru5P for use in NMR experiments and shown enzymatic formation of DHBP and formate. We also show the 1.1 Å structure of apo RibB. Previously reported product bound structures of RibB have been modeled with two metal ions in the active site2. However, we report here intrinsic tyrosine fluorescence and activity assay profiles that indicate that RibB requires a single magnesium for full activity.Support or Funding InformationWeaver Graduate Student Fellowship, the National Science Foundation CHE‐1403293, The University of Kansas 2018 General Research FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.