Identification of the First Riboflavin Catabolic Gene Cluster Isolated from Microbacterium maritypicum G10

Abstract

Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed.Cofactors play key roles in augmenting the limited functionality available on proteins for catalysis. As with all other metabolites, cofactors do not accumulate in the environment. This is due in part to efficient salvage of these biosynthetically expensive metabolites and in part to cofactor catabolism. However, in contrast to the large literature on cofactor biosynthesis, relatively little is known about cofactor breakdown (1). Detailed enzymology has only been carried out on heme (2), pyridoxal (3), and NAD (4, 5) catabolism. Thiamin, folate, riboflavin, and biotin degrading bacteria were once isolated but have now been lost (6 -11). Analysis of culture metabolites from the reported riboflavin catabolic strain demonstrated that this strain was capable of fully degrading the isoalloxazine ring of riboflavin (12). None of the genes were identified and lumichrome was not an intermediate. Riboflavin hydrolase, the only previously characterized riboflavin catabolic enzyme, catalyzes the conversion of riboflavin to lumichrome and ribose and has previously been identified as a potential catabolic enzyme in a second pathway (13-15). However, despite significant effort, the gene encoding this enzyme was never identified and the enzyme itself proved to be biochemically intractable (16). Here we report the isolation of a riboflavin-catabolizing Microbacterium maritypicum strain from dust samples obtained at the DSM riboflavin production plant in Germany. The catabolic operon was isolated from a cosmid library, sequenced, and annotated. Preliminary characterization of the enzymes involved suggested a riboflavin catabolic pathway. ResultsStrain M. maritypicum G10 Catabolizes Riboflavin-Dust samples, collected at the DSM riboflavin production plant in Germany, were screened by culturing in a defined minimal medium containing M9 salts with riboflavin as the primary carbon source. Bleaching of the riboflavin color was observed in some of the samples. The cells from these hits were grown on NB plates and single colonies were selected. Individual strains were retested in M9 medium with riboflavin as the sole carbon source. The best of these strains (strain G10) completely bleached the yellow color of riboflavin after 12 h of growth and was selected for further study. The sequence of the gene encoding 16S rRNA identified this strain as M. maritypicum.To identify the products generated from consumption of riboflavin, M. maritypicum G10 was gro...

Two-Component Flavin-Dependent Riboflavin Monooxygenase Degrades Riboflavin in Devosia riboflavina

“…These results agreed with the notion that D. riboflavina degraded riboflavin to lumichrome and D-ribose, the latter of which is a bacterial source of carbon and energy. Notably, this notion could revise the proposal by Yanagita and Foster that bacterial riboflavin is degraded to ribitol (15), but it is consistent with the proposal that the M. maritypicum G10 mechanism generates D-ribose (17). We then further investigated the mechanism of riboflavin degradation by D. riboflavina.…”

“…Monooxygenation of riboflavin to lumichrome and D-ribose. We investigated the reaction by D. riboflavina rFMO in detail because the M. maritypicum RcaE protein, which is an oxygen-dependent hydrolase, is unlikely because the M. maritypicum RcaE product is D-ribose (17), which would not be a simple hydrolase product. The native FMN spectrum has absorption peaks at 371 nm and 444 nm.…”

“…Microbacteria and related Actinomycetales bacteria (17). Since D. riboflavina FMO has only 67% amino acid identity with M. maritypicum FMO (GenBank accession number WP_021201082.1), we revisited the phylogenetic analysis of those proteins.…”

“…The molecular mechanism of riboflavin degradation has remained obscure for 70 years. However, the actinobacterium Microbacterium maritypicum G10 was recently isolated from a riboflavin production plant, and the rca gene cluster for bacterial riboflavin catabolism was identified, although nucleotide sequences were not published (17). The RcaE protein encoded by the gene cluster is thought to be riboflavin hydrolase, but the reaction between recombinant RcaE and riboflavin produced only D-ribose in addition to lumichrome, indicating that this is not a simple hydrolase reaction that produces ribitol (15).…”

Increased algicidal activity of Aeromonas veronii in response to Microcystis aeruginosa: interspecies crosstalk and secondary metabolites synergism

“…The enzymatic reactions leading to lumichrome biosynthesis were characterized as early as 1956 (Yanagita and Foster, 1956), but there is little information about the enzyme itself. Recent studies implicated riboflavin monooxygenase (67% identical to RcaE that functions as hydrolase) in riboflavin degradation in α-proteobacteria (Kanazawa et al, 2018), whereas riboflavin hydrolase was implicated in a microbacterium (Xu et al, 2016). The biological role of lumichrome is not clear but it was implicated as an inhibitor of quorum sensing systems (Park et al, 2006;Li et al, 2011a;Abed et al, 2013).…”