RosB from Streptomyces davawensis is the key enzyme of roseoflavin biosynthesis

Abstract

The antibiotic roseoflavin (RoF) is the only known natural riboflavin (vitamin B2) analogue and is active against gram-positive bacteria. RoF is produced by Streptomyces cinnabarinus (S. cinnabarinus) and Streptomyces davawensis (S. davawensis) and can be considered to be an “antivitamin”. In RoF biosynthesis one of the methyl groups of the predicted precursor riboflavin undergoes a site-specific replacement by a dimethyl amino group whereby 8-demethyl-8-amino-riboflavin (AF) was postulated to be an intermediate. The first discovered enzyme of roseoflavin biosynthesis was the S-adenosyl methionine (SAM) dependent dimethyltransferase RosA which converts AF to RoF. Subsequent systematic gene deletion experiments carried out in the RoF-producer S. davawensis suggested that a single enzyme (RosB) is responsible for the formation of AF. However, when recombinant RosB was tested in an assay mixture containing riboflavin-5’-phosphate (RP) the formation of the predicted final reaction product AF was not observed. Instead the compound 8-demethyl-8-formyl-riboflavin-5’-phosphate (HOC-RP) was detected, probably an intermediate of the RosB reaction. How the formyl-group of HOC-RP was replaced by an amino group to give AF or 8-demethyl-8-amino-riboflavin-5’-phosphate (AFP) remained unclear. The present work was initiated to investigate the predicted oxidation of HOC-RP to 8-demethyl-8-carboxyl-riboflavin-5’-phosphate (HOOC-RP), to identify the amino group donor of the RosB reaction and to shed light on the reaction mechanism of the multi-step enzyme RosB. It was found that RosB accepts only RP as a substrate and not riboflavin (RF). RosB activity depends on the presence of O2, thiamine and the amino group donor glutamate. HOOC-RP was found to be an (additional) intermediate of the RosB reaction. The crystal structure of RosB was solved with bound AFP (1.7 A) and HOC-RP (2.0 A). RosB is composed of four flavodoxin-like subunits which have been upgraded with specific extensions and a unique C-terminal arm. Structure-based active site analysis was complemented by mutational and isotope-based mass-spectrometric data to propose an enzymatic mechanism. The present work also shows that the RoF biosynthetic pathway still has not been completely resolved. RosB releases AFP, yet the substrate for the subsequent RosA reaction is AF. Consequently, a phosphatase must be present which has not yet been identified.