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-A 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.
Highlights
The most frequently used cofactors in enzymatic redox reactions are the pyridine (NAD and NADP) and the flavin (FAD and FMN) nucleotides
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
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
Summary
Cloning and Purification—The YLR011w open reading frame DNA was amplified by PCR using oligodeoxynucleotides synthesized by MWG Biotech and cloned between the NdeI and NotI sites of pET29 vector from Novagen. Crystallization and Resolution of the Structure—Native and Se-Met labeled protein samples were stored in 20 mM Tris-HCl, pH 8, 100 mM NaCl, and 20 mM -mercaptoethanol. 20 mM CaCl2 and 2 mM FMN were added to the protein solutions Crystals of both the native and Se-Met-labeled protein were grown from a 1:1-l mixture of protein (6 mg/ml) with 10 –15% polyethylene glycol 3000, 20% polyethylene glycol 400, and 0.1 M Tris-HCl, pH 8.5 (reservoir solution). Azoreductase assay was performed in a reaction mixture (1 ml) containing 35 M azo dye, 100 M NADH or NADPH, 20 M FMN, and 2.4 M of enzyme in 25 mM Tris-HCl, pH 7.5. Se-Met, selenium Met; r.m.s.d., root mean square deviation
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