This entry represents the FMN-binding domain found in NAD(P)H-flavin oxidoreductases (flavin reductases), a class of enzymes capable of producing reduced flavin for bacterial bioluminescence and other biological processes. This domain is also found in various other oxidoreductase and monooxygenase enzymes (PUBMED:12829278), (PUBMED:15461461), (PUBMED:11017201). This domain consists of a beta-barrel with Greek key topology, and is related to the ferredoxin reductase-like FAD-binding domain. The flavin reductases have a different dimerisation mode than that found in the PNP oxidase-like family, which also carries an FMN-binding domain with a similar topology.
The FMN-binding domain is found in NAD(P)H-flavin oxidoreductases (flavin reductases), a class of enzymes capable of producing reduced flavin for bacterial bioluminescence and other biological processes, and various other oxidoreductase and monooxygenase enzymes [(PUBMED:12829278), (PUBMED:15461461), (PUBMED:11017201)].
This domain consists of a beta-barrel with Greek key topology, and is related to the ferredoxin reductase-like FAD-binding domain. The flavin reductases have a different dimerisation mode than that found in the PNP oxidase-like family, which also carries an FMN-binding domain with a similar topology.
Aminobacter aminovorans NADH:flavin oxidoreductase His140: a highlyconserved residue critical for NADH binding and utilization.
Biochemistry. 2004; 43: 12887-93
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Homodimeric FRD(Aa) Class I is an NADH:flavin oxidoreductase fromAminobacter aminovorans. It is unusual because it contains an FMN cofactorbut utilizes a sequential-ordered kinetic mechanism. Because little isknown about NADH-specific flavin reductases in general and FRD(Aa) inparticular, this study aimed to further explore FRD(Aa) by identifying thefunctionalities of a key residue. A sequence alignment of FRD(Aa) withseveral known and hypothetical flavoproteins in the same subfamily revealswithin the flavin reductase active-site domain a conserved GDH motif,which is believed to be responsible for the enzyme and NADH interaction.Mutation of the His140 in this GDH motif to alanine reduced FRD(Aa)activity to <3%. An ultrafiltration assay and fluorescence quenchingdemonstrated that H140A FRD(Aa) binds FMN in the same 1:1 stoichiometricratio as the wild-type enzyme, but with slightly weakened affinity (K(d) =0.9 microM). Anaerobic stopped-flow studies were carried out using boththe native and mutated FRD(Aa). Similar to the native enzyme, H140AFRD(Aa) was also able to reduce the FMN cofactor by NADH although muchless efficiently. Kinetic analysis of anaerobic reduction measurementsindicated that the His140 residue of FRD(Aa) was essential to NADHbinding, as well as important for the reduction of the FMN cofactor. Forthe native enzyme, the cofactor reduction was followed by at least oneslower step in the catalytic pathway.
Almost all organisms require iron for enzymes involved in essentialcellular reactions. Aerobic microbes living at neutral or alkaline pHencounter poor iron availability due to the insolubility of ferric iron.Assimilatory ferric reductases are essential components of the ironassimilatory pathway that generate the more soluble ferrous iron, which isthen incorporated into cellular proteins. Dissimilatory ferric reductasesare essential terminal reductases of the iron respiratory pathway iniron-reducing bacteria. While our understanding of dissimilatory ferricreductases is still limited, it is clear that these enzymes are distinctfrom the assimilatory-type ferric reductases. Research over the last 10years has revealed that most bacterial assimilatory ferric reductases areflavin reductases, which can serve several physiological roles. Thisarticle reviews the physiological function and structure of assimilatoryand dissimilatory ferric reductases present in the Bacteria, Archaea andYeast. Ferric reductases do not form a single family, but appear to bedistinct enzymes suggesting that several independent strategies for ironreduction may have evolved.
A set of 424 nonmembrane proteins from Methanobacteriumthermoautotrophicum were cloned, expressed and purified for structuralstudies. Of these, approximately 20% were found to be suitable candidatesfor X-ray crystallographic or NMR spectroscopic analysis without furtheroptimization of conditions, providing an estimate of the number of themost accessible structural targets in the proteome. A retrospectiveanalysis of the experimental behavior of these proteins suggested somesimple relations between sequence and solubility, implying that data basesof protein properties will be useful in optimizing high throughputstrategies. Of the first 10 structures determined, several provided cluesto biochemical functions that were not detectable from sequence analysis,and in many cases these putative functions could be readily confirmed bybiochemical methods. This demonstrates that structural proteomics isfeasible and can play a central role in functional genomics.
An NADPH:FAD oxidoreductase from the valanimycin producer, Streptomycesviridifaciens. Cloning, analysis, and overexpression.
J Biol Chem. 1997; 272: 23303-11
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The valanimycin producer Streptomyces viridifaciens contains atwo-component enzyme system that catalyzes the oxidation of isobutylamineto isobutylhydroxylamine. One component of this enzyme system isisobutylamine hydroxylase, and the other component is a flavin reductase.The gene (vlmR) encoding the flavin reductase required by isobutylaminehydroxylase has been cloned from S. viridifaciens by chromosome walking.The gene codes for a protein of 194 amino acids with a calculated mass of21,265 Da and a calculated pI of 10.2. Overexpression of the vlmR gene inEscherichia coli as an N-terminal His-tag derivative yielded a solubleprotein that was purified to homogeneity. Removal of the N-terminalHis-tag from the overexpressed protein by thrombin cleavage also produceda soluble protein. Both forms of the protein exhibited a high degree offlavin reductase activity, and the thrombin-cleaved form functioned incombination with isobutylamine hydroxylase to catalyze the conversion ofisobutylamine to isobutylhydroxylamine. Kinetic data indicate that theoverexpressed protein utilizes FAD and NADPH in preference to FMN,riboflavin, and NADH. The deduced amino acid sequence of the VlmR proteinexhibited similarity to several other flavin reductases that mayconstitute a new family of flavin reductases.
Cloning and analysis of structural genes from Streptomycespristinaespiralis encoding enzymes involved in the conversion ofpristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase andNADH:riboflavin 5'-phosphate oxidoreductase.
J Bacteriol. 1995; 177: 5206-14
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In Streptomyces pristinaespiralis, two enzymes are necessary forconversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), themajor component of pristinamycin (D. Thibaut, N. Ratet, D. Bisch, D.Faucher, L. Debussche, and F. Blanche, J. Bacteriol. 177:5199-5205, 1995);these enzymes are PIIA synthase, a heterodimer composed of the SnaA andSnaB proteins, which catalyzes the oxidation of PIIB to PIIA, and theNADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMNreductase), the SnaC protein, which provides the reduced form of flavinmononucleotide for the reaction. By using oligonucleotide probes designedfrom limited peptide sequence information of the purified proteins, thecorresponding genes were cloned from a genomic library of S.pristinaespiralis. SnaA and SnaB showed no significant similarity withproteins from databases, but SnaA and SnaB had similar protein domains.Disruption of the snaA gene in S. pristinaespiralis led to accumulation ofPIIB. Complementation of a S. pristinaespiralis PIIA-PIIB+ mutant with thesnaA and snaB genes, cloned in a low-copy-number plasmid, partiallyrestored production of PIIA. The deduced amino acid sequence of the snaCgene showed no similarity to the sequences of other FMN reductases but was39% identical with the product of the actVB gene of the actinorhodincluster of Streptomyces coelicolor A(3)2, likely to be involved in thedimerization step of actinorhodin biosynthesis. Furthermore, an S.coelicolor A(3)2 mutant blocked in this step was successfully complementedby the snaC gene, restoring the production of actinorhodin.
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