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Identification of the riboflavin cofactor-binding site in the Vibrio cholerae ion-pumping NQR complex: A novel structural motif in redox enzymes.

ABSTRACT: The ion-pumping NQR complex is an essential respiratory enzyme in the physiology of many pathogenic bacteria. This enzyme transfers electrons from NADH to ubiquinone through several cofactors, including riboflavin (vitamin B2). NQR is the only enzyme reported that is able to use riboflavin as a cofactor. Moreover, the riboflavin molecule is found as a stable neutral semiquinone radical. The otherwise highly reactive unpaired electron is stabilized via an unknown mechanism. Crystallographic data suggested that riboflavin might be found in a superficially located site in the interface of NQR subunits B and E. However, this location is highly problematic, as the site does not have the expected physiochemical properties. In this work, we have located the riboflavin-binding site in an amphipathic pocket in subunit B, previously proposed to be the entry site of sodium. Here, we show that this site contains absolutely conserved residues, including N200, N203, and D346. Mutations of these residues decrease enzymatic activity and specifically block the ability of NQR to bind riboflavin. Docking analysis and molecular dynamics simulations indicate that these residues participate directly in riboflavin binding, establishing hydrogen bonds that stabilize the cofactor in the site. We conclude that riboflavin is likely bound in the proposed pocket, which is consistent with enzymatic characterizations, thermodynamic studies, and distance between cofactors.

SUBMITTER: Tuz K

PROVIDER: S-EPMC9293633 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Riboflavin is the precursor of important redox cofactors such as flavin mononucleotide (FMN) and flavin adenine dinucleotide, required for several biological processes. Vibrio cholerae, a pathogenic bacterium responsible for the cholera disease, possesses the ability to biosynthesize de novo as well as to uptake riboflavin through the riboflavin biosynthetic pathway (RBP) and the RibN importer, respectively. The intra-organism relationship between riboflavin biosynthesis and uptake functions has not been studied. RESULTS:This work determined the transcriptional organization of RBP genes and ribN in V. cholerae through reverse transcription polymerase chain reaction and analyzed their expression when growing with or without extracellular riboflavin using real time PCR. The RBP is organized in three transcriptional units, the major one containing ribD, ribE, ribA and ribH together with genes involved in functions not directly related to riboflavin biosynthesis such as nrdR and nusB. In addition, two independent monocistronic units contain ribA2 and ribB, the later conserving a putative FMN riboswitch. The ribN gene is encoded in operon with a gene coding for a predicted outer membrane protein and a gene encoding a protein with a glutaredoxin domain. Regulation analysis showed that among these transcriptional units, only ribB is negatively regulated by riboflavin and that its repression depends on the RibN riboflavin importer. Moreover, external riboflavin highly induced ribB transcription in a ?ribN strain. Also, a genomic database search found a negative correlation between the presence of nrdR and nusB and the FMN riboswitch in bacterial RBP operons. CONCLUSIONS:Growing in the presence of riboflavin downregulates only a single element among the transcriptional units of riboflavin supply pathways. Thus, endogenous riboflavin biosynthesis seems to be negatively regulated by extracellular riboflavin through its specific effect on transcription of ribB in V. cholerae.

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Identification of the riboflavin cofactor-binding site in the Vibrio cholerae ion-pumping NQR complex: A novel structural motif in redox enzymes.

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