Crystal Structure and Functional Characterization of Yeast YLR011wp, an Enzyme with NAD(P)H-FMN and Ferric Iron Reductase Activities

Abstract

Flavodoxins are involved in a variety of electron transfer reactions that are essential for life. Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins. We describe the 2.0-Å resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein. YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer. Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins. YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one. Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp. OY1-2 azoreductase. We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity. We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds.The most frequently used cofactors in enzymatic redox reactions are the pyridine (NAD and NADP) and the flavin (FAD and FMN) nucleotides. Although NAD and NADP are soluble cofactors used by dehydrogenases, FAD and FMN usually work as prosthetic groups in flavoproteins to which they are tightly bound. Flavodoxins are small monomeric flavoproteins (15-22 kDa) that noncovalently bind a single FMN molecule, acting as a redox center (1). The flavodoxin scaffold contributes to the mechanism of electron transfer by stabilizing the FMN molecule in an environment that promotes the highly negative redox potential required for its biochemical activity. This is accomplished by sandwiching the FMN ring between two hydrophobic side chains. Flavodoxins are involved in a variety of electron transfer reactions that are essential in the metabolism of pyruvate (2), nitrogen, and pyridine nucleotides (3). These enzymes exist under three redox states: oxidized; partially reduced (semiquinone); and fully reduced (hydroquinone). Until now, flavodoxins have been identified in many prokaryotes and also in some eukaryotic algae (4 -6). In mammalian systems, flavodoxins have only been found as domains inserted in larger redox proteins such as cytochrome P450 reductase where they act as the electron transport intermediate between FAD and the P450 iron center (7). Flavodoxin-like domains are also found in mammalian nitric-oxide synthase (8) and in human erythrocyte NADPH-flavin reductase (9).The Saccharomyces cerevisiae YLR011w open reading frame codes for a protein of unknown function that has been found to be up-regulated in response to low temperature exposure (10). The sequence belongs to a COG4530 (Cluster of Orthologous Genes) predicted to generally contain flavoproteins. Hence, the YLR011wp 21-kDa protein was proposed to be composed of a single domain present in NADPH-dependent FMN reductases. We have solved the 2-Å resolution crystal structure, ...

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

“…ecAzoR, rsAzoR, stAzoR and efAzoR are azoreductases from E. coli (Nakanishi et al, 2001), R. sphaeroides (Bin et al, 2004), S. typhimurium (Zhang et al, 2004) and E. faecalis (Chen et al, 2004). paWrbA is the sequence for WrbA from P. aeruginosa (Gorman and Shapiro, 2005), KefF is an enzyme from E. coli (Roosild et al, 2009), ecMdaB and paMdaB are the mediator of drug activity B from E. coli (Adams and Jia, 2006) and P. aeruginosa respectively, YhdA is an enzyme from B. subtilis (Binter et al, 2009), EmoB is an enzyme fromMesorhizobiums p. BNC1 (Nissen et al, 2008), ArsH is an enzyme from S. meliloti (Ye et al, 2007), Lot6p is an enzyme from S. cerevisiae (Liger et al, 2004), ppChrR is the chromate reductase from P. putida (Ackerley et al, 2004). 1X77 is an NADPH dependent oxidoreductase from P. aeruginosa (Agarwal et al, 2006), hNQO1 and hNQO2 are human NQOs (Foster et al, 1999;Li et al, 1995), A. tha is the sequence for an NAD(P)H quinone reductase from A. thaliana (Sparla et al, 1999).…”

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations

“…In this mechanism, two cycles of NADPH-dependent reduction of FMN to FMNH occur, converting azo dye to a hydrazine in the first step, and then the hydrazine to two constitutive amines in the second step (Correia et al, 2011;Ryan et al, 2010). Flavin dependent azoreductases are polymeric in nature, and flavin molecules are non-covalently attached with protein and link the dimeric sub units of azoreductases (Liger et al, 2004). Flavin contributes thermal stability to azoreductase enzyme (Natalello et al, 2007).…”

Three-dimensional Structure of AzoR from Escherichia coli

“…deviation ϭ 2.4 Å for 142 residues of a total 165 residues), although its function has not yet been documented, has a structure similar to that of yeast YLR011wp (accession code 1T0I; Z score ϭ 19.4 and r.m.s. deviation ϭ 2.3 Å for 156 residues of a total 191 residues, compared with 1NNI), and YLR001wp was characterized as an FMN-dependent NAD(P)H:ferric iron oxidoreductase (42). All of these flavoenzymes exist as homodimers.…”