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Abstract

Roseoflavin is an analogue of riboflavin (Vitamin B2) and shows antimicrobial activities against various Gram-positive bacteria. Roseoflavin targets flavin mononucleotide (FMN) riboswitches and flavoenzymes that require FMN and flavin adenine dinucleotide (FAD) as cofactors, thus negatively affecting riboflavin uptake, riboflavin biosynthesis and cellular metabolism. The roseoflavin biosynthesis pathway converts FMN to roseoflavin using multiple steps catalysed by the enzymes, RosB, RosC and RosA. This study focuses on the enzyme RosC, which catalyses the dephosphorylation of 8-demethyl-8-amino-riboflavin-5ʹ-phosphate (AFP) to 8-demethyl-8-amino-riboflavin (AF) which both are intermediates of roseoflavin biosynthesis. RosC belongs to the histidine phosphatase superfamily. Dephosphorylation activity was observed for AFP and FMN, which are structurally similar compounds, and dephosphorylation of FMN, in combination with an FMN-producing flavokinase, in principle, would generate a futile cycle. RosC was purified to apparent homogeneity from a recombinant Escherichia coli strain. Such a preparation was used to set up crystal screens and four different crystal structures were obtained. Crystal structures of flavin-free RosC, RosC in complex with AF (1.7 Å), RosC in complex with AF (1.25 Å) and inorganic phosphate (1.55 Å, mimicking the AFP bound state) and the RosC variant RosCR33A in complex with riboflavin (1.5 Å) were complemented with comprehensive mutational/kinetic studies. Extensive structural and functional studies on this protein indicated the presence of highly specialized and unique segments and arrangements of residues that assist in binding the substrate. Substrate-induced rigidification of the elongated strand-to-helix insertions and the helical N-terminal extension promote substrate binding and ensure substrate specificity. Aromatic residues in the binding pocket mediate stacking interactions with the isoalloxazine ring of the bound flavin. The amino acid, D166 of RosC, was identified as the critical residue that discriminates between the flavin substrates AFP and FMN. D166 binds the isoalloxazine and ribityl groups of AF, and site-specific exchanges at this position drastically reduce the catalytic activity of RosC. Mutations of D166 to hydrophobic residues (valine, isoleucine and leucine) impart toxic properties to RosC when overproduced in Escherichia coli and Corynebacterium glutamicum but not when overproduced in Bacillus subtilis. The reason for this remains unknown. Two other FMN dephosphorylating enzymes in S. davaonensis were identified and characterized by gene expression studies.