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.
Highlights
Cofactors play key roles in augmenting the limited functionality available on proteins for catalysis
We report the isolation of a riboflavin-catabolizing Microbacterium maritypicum strain from dust samples obtained at the DSM riboflavin production plant in Germany
The purified enzyme was incubated with riboflavin and ATP and the product was identified as FMN by HPLC co-elution with the purchased standard, confirming RcaA as a riboflavin kinase (Fig. 3B)
Summary
Strain 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. 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. Based on an estimated Microbacterium genome size of 4 –5 Mbp, we constructed a library of 768 clones (eight 96-well plates) resulting in at least 6-fold coverage of the genome This library was screened, first in batches of 12 cosmids, individually, to yield two cosmids encoding the riboflavin catabolic pathway (1A4 and 5H7, Fig. 2A). Cosmids 5H7 and 1A4 both contain all of the genes essential for riboflavin catabolism in S. lividans (supplemental Table S2). The purified enzyme was incubated with riboflavin and ATP and the product was identified as FMN by HPLC co-elution with the purchased standard, confirming RcaA as a riboflavin kinase (Fig. 3B). The overexpressed enzyme was purified on an amylose resin column followed by maltose tag
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