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Structure-Guided Analyses of a Key Enzyme Involved in the Biosynthesis of an Antivitamin.

RosB catalyzes the formation of 8-aminoriboflavin 5'-phosphate (AFP), the key intermediate in roseoflavin biosynthesis, from the metabolic precursors riboflavin 5'-phosphate (RP, also known as FMN) and glutamate. The conversion of the aromatic methyl group at position 8 in RP into the aromatic amine in AFP occurs via two intermediates, namely, the aldehyde 8-formyl-RP and the acid 8-carboxy-RP. To gain insights into the mechanism for this chemically challenging transformation, we utilized a structure-based approach to identify active site variants of RosB that stall the reaction at various points along the reaction coordinate. Crystal structures of individual variants in complex with different reaction intermediates, identified via mass spectroscopic analysis, illuminate conformational changes that occur at the active site during multistep conversion. These studies provide a plausible route for the progression of the reaction and a molecular rationale for the mechanism of this unusual biocatalyst.