ABSTRACT
Flavins are ubiquitous molecules in life as they serve as important enzyme cofactors. In the Gram-positive, soil-dwelling bacterium Bacillus subtilis, four well-characterized gene products (the enzymes RibDG, RibE, RibAB, and RibH) catalyze the biosynthesis of riboflavin (RF) from guanosine-triphosphate (GTP) and ribulose-5-phosphate (R5P). The corresponding genes form an operon together with the gene ribT (ribDG-E-AB-H-T), wherein the function of this terminal gene remained enigmatic. RibT has been structurally characterized as a GCN5-like acetyltransferase (GNAT), however, with unidentified target molecules. Bacterial two-hybrid system revealed interactions between RibT, RibH, and RibE, forming the heavy RF synthase complex. Applying single particle tracking (SPT), we found that confined (sub)diffusion of RibT is largely dependent on interacting RibE and, to a lesser degree, on interacting RibH. By induced expression of otherwise low-expressed ribT from an ectopic locus, we observed a decrease in the subpopulation considered to represent capsids of the heavy RF synthase and an increase in the subpopulation thought to represent pentamers of RibH, pointing to a putative role for RibT in capsid disassembly. Complementarily, either deletion of ribT or mutation of a key residue from RibH (K29) suspected to be the substrate of RibT for acetylation leads to increased levels of subpopulations considered as capsids of RibH-mVenus (RibH-mV) in comparison to wild-type (wt)-like cells. Thus, we provide evidence for an indirect involvement of RibT in RF biosynthesis by a putative capsid disassembling mechanism considered to involve acetylation of RibH residue K29 at the three-fold symmetry axis of 60-mer capsids.
ABSTRACT
Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for biosynthesis and/or transport of riboflavin (rib genes). Cytoplasmic riboflavin is rapidly and almost completely converted to FMN by flavokinases. When cytoplasmic levels of FMN are sufficient ("high levels"), FMN binding to FMN riboswitches leads to a reduction of rib gene expression. We report here that the protein RibR counteracts the FMN-induced "turn-off" activities of both FMN riboswitches in Bacillus subtilis, allowing rib gene expression even in the presence of high levels of FMN. The reason for this secondary metabolic control by RibR is to couple sulfur metabolism with riboflavin metabolism.
Subject(s)
Bacillus subtilis/metabolism , Flavin Mononucleotide/metabolism , Gene Expression Regulation, Bacterial/genetics , Riboflavin/metabolism , Riboswitch/physiology , Sulfur/metabolism , Electrophoretic Mobility Shift Assay , Metabolome/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Streptomyces davawensis synthesizes the antibiotic roseoflavin (RoF) (8-dimethylamino-8-demethyl-D-riboflavin). It was postulated that RoF is synthesized from riboflavin via 8-amino- (AF) and 8-methylamino-8-demethyl-D-riboflavin (MAF). In a cell-free extract of S. davawensis, an S-adenosyl methionine-dependent conversion of AF into MAF and RoF was observed. The corresponding N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity was enriched by column chromatography. The final most active fraction still contained at least five different proteins that were analyzed by enzymatic digestion and concomitant de novo sequencing by MS/MS. One of the sequences matched a hypothetical peptide fragment derived from an as yet uncharacterized open reading frame (sda77220) located in the middle of a (putative) gene cluster within the S. davawensis genome. Expression of ORF sda77220 in Escherichia coli revealed that the corresponding gene product had N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity. Inactivation of ORF sda77220 led to a S. davawensis strain that synthesized AF but not MAF or RoF. Accordingly, as the first identified gene of RoF biosynthesis, ORF sda77220 was named rosA. RosA (347 amino acids; 38 kDa) was purified from a recombinant E. coli strain (as a His(6)-tagged protein) and was biochemically characterized (apparent K(m) for AF = 57.7 ± 9.2 µm; apparent K(D) for AF = 10.0 µm; k(cat) = 0.37 ± 0.02 s(-1)). RosA is a unique enzyme and may be useful for a variety of applications.