Dataset Information

Crystal structures of the lumazine protein from Photobacterium kishitanii in complexes with the authentic chromophore, 6,7-dimethyl- 8-(1'-D-ribityl) lumazine, and its analogues, riboflavin and flavin mononucleotide, at high resolution.

ABSTRACT: Lumazine protein (LumP) is a fluorescent accessory protein having 6,7-dimethyl-8-(1'-d-ribityl) lumazine (DMRL) as its authentic chromophore. It modulates the emission of bacterial luciferase to shorter wavelengths with increasing luminous strength. To obtain structural information on the native structure as well as the interaction with bacterial luciferase, we have determined the crystal structures of LumP from Photobacterium kishitanii in complexes with DMRL and its analogues, riboflavin (RBF) and flavin mononucleotide (FMN), at resolutions of 2.00, 1.42, and 2.00 A. LumP consists of two beta barrels that have nearly identical folds, the N-terminal and C-terminal barrels. The structures of LumP in complex with all of the chromophores studied are all essentially identical, except around the chromophores. In all of the structures, the chromophore is tethered to the narrow cavity via many hydrogen bonds in the N-terminal domain. These are absent in the C-terminal domain. Hydrogen bonding in LumP-FMN is decreased in comparison with that in LumP-RBF because the phosphate moiety of FMN protrudes out of the narrow cavity. In LumP-DMRL, the side chain of Gln65 is close to the ring system, and a new water molecule that stabilizes the ligand is observed near Ser48. Therefore, DMRL packs more tightly in the ligand-binding site than RBF or FMN. A docking simulation of bacterial luciferase and LumP suggests that the chromophore is located close enough for direct energy transfer to occur. Moreover, the surface potentials around the ligand-binding sites of LumP and bacterial luciferase exhibit complementary charge distributions, which would have a significant effect on the interaction between LumP and luciferase.

SUBMITTER: Sato Y

PROVIDER: S-EPMC2798275 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Crystal structures of the lumazine protein from Photobacterium kishitanii in complexes with the authentic chromophore, 6,7-dimethyl- 8-(1'-D-ribityl) lumazine, and its analogues, riboflavin and flavin mononucleotide, at high resolution.

Sato Yuichi Y Shimizu Satoshi S Ohtaki Akashi A Noguchi Keiichi K Miyatake Hideyuki H Dohmae Naoshi N Sasaki Satoshi S Odaka Masafumi M Yohda Masafumi M

Journal of bacteriology 20100101 1

Lumazine protein (LumP) is a fluorescent accessory protein having 6,7-dimethyl-8-(1'-d-ribityl) lumazine (DMRL) as its authentic chromophore. It modulates the emission of bacterial luciferase to shorter wavelengths with increasing luminous strength. To obtain structural information on the native structure as well as the interaction with bacterial luciferase, we have determined the crystal structures of LumP from Photobacterium kishitanii in complexes with DMRL and its analogues, riboflavin (RBF) ...[more]

PMID: 19854891

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Project description:The actinobacterium Microbacterium maritypicum splits riboflavin (vitamin B2) into lumichrome and d-ribose. However, such degradation by other bacteria and the involvement of a two-component flavin-dependent monooxygenase (FMO) in the reaction remain unknown. Here we investigated the mechanism of riboflavin degradation by the riboflavin-assimilating alphaproteobacterium Devosia riboflavina (formerly Pseudomonas riboflavina). We found that adding riboflavin to bacterial cultures induced riboflavin-degrading activity and a protein of the FMO family that had 67% amino acid identity with the predicted riboflavin hydrolase (RcaE) of M. maritypicum MF109. The D. riboflavina genome clustered genes encoding the predicted FMO, flavin reductase (FR), ribokinase, and flavokinase, and riboflavin induced their expression. This finding suggests that these genes constitute a mechanism for utilizing riboflavin as a carbon source. Recombinant FMO (rFMO) protein of D. riboflavina oxidized riboflavin in the presence of reduced flavin mononucleotide (FMN) provided by recombinant FR (rFR), oxidized FMN and NADH, and produced stoichiometric amounts of lumichrome and d-ribose. Further investigation of the enzymatic properties of D. riboflavina rFMO indicated that rFMO-rFR coupling accompanied O2 consumption and the generation of enzyme-bound hydroperoxy-FMN, which are characteristic of two-component FMOs. These results suggest that D. riboflavina FMO is involved in hydroperoxy-FMN-dependent mechanisms to oxygenize riboflavin and a riboflavin monooxygenase is necessary for the initial step of riboflavin degradation.IMPORTANCE Whether bacteria utilize either a monooxygenase or a hydrolase for riboflavin degradation has remained obscure. The present study found that a novel riboflavin monooxygenase, not riboflavin hydrolase, facilitated this process in D. riboflavina The riboflavin monooxygenase gene was clustered with flavin reductase, flavokinase, and ribokinase genes, and riboflavin induced their expression and riboflavin-degrading activity. The gene cluster is uniquely distributed in Devosia species and actinobacteria, which have exploited an environmental niche by developing adaptive mechanisms for riboflavin utilization.

Project description:Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s-1 Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s-1 The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes.IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.

Dataset Information

Crystal structures of the lumazine protein from Photobacterium kishitanii in complexes with the authentic chromophore, 6,7-dimethyl- 8-(1'-D-ribityl) lumazine, and its analogues, riboflavin and flavin mononucleotide, at high resolution.

Publications

Crystal structures of the lumazine protein from Photobacterium kishitanii in complexes with the authentic chromophore, 6,7-dimethyl- 8-(1'-D-ribityl) lumazine, and its analogues, riboflavin and flavin mononucleotide, at high resolution.

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