The ribR gene encodes a monofunctional riboflavin kinase which is involved in regulation of the Bacillus subtilis riboflavin operon

A 3.5 kb EcoRI-BamHI fragment of Bacillus subtilis chromosomal DNA carrying the ribR gene, involved in regulation of the B. subtilis riboflavin operon, was cloned in the B. subtilis-Escherichia coli shuttle vector pCB20. DNA sequence analysis of this fragment revealed several ORFs, one of which encodes a polypeptide of 230 amino acids with up to 45% sequence identity with FAD synthetases from a number of micro-organisms, such as Corynebacterium ammoniagenes, E. coli and Pseudomonas fluorescens, and also to the ribC gene product of B. subtilis. The ribR gene was amplified by PCR, cloned and expressed in E. coli. Measurement of flavokinase activity in cell extracts demonstrated that ribR encodes a monofunctional flavokinase which converts riboflavin into FMN but not to FAD, and is specific for the reduced form of riboflavin.