Crystal structure of a new type of NADPH-dependent quinone oxidoreductase (QOR2) from Escherichia coli.
ABSTRACT Escherichia coli QOR2 [NAD(P)H-dependent quinone oxidoreductase; a ytfG gene product], which catalyzes two-electron reduction of methyl-1,4-benzoquinone, is a new type of quinone-reducing enzyme with distinct primary sequence and oligomeric conformation from previously known quinone oxidoreductases. The crystal structures of native QOR2 and the QOR2-NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) complex reveal that QOR2 consists of two domains (N-domain and C-domain) resembling those of NmrA, a negative transcriptional regulator that belongs to the short-chain dehydrogenase/reductase family. The N-domain, which adopts the Rossmann fold, provides a platform for NADPH binding, whereas the C-domain, which contains a hydrophobic pocket connected to the NADPH-binding site, appears to play important roles in substrate binding. Asn143 near the NADPH-binding site has been identified to be involved in substrate binding and catalysis from structural and mutational analyses. Moreover, compared with wild-type strain, the qor2-overexpressing strain shows growth retardation and remarkable decrease in several enzymes involved in carbon metabolism, suggesting that QOR2 could play some physiological roles in addition to quinone reduction.
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ABSTRACT: The respiratory chain of Escherichia coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E. coli accept electrons from this naphtoquinone derivative.FEBS Journal 04/2012; 279(18):3364-73. · 4.25 Impact Factor
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ABSTRACT: Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal reduction, the enzyme kinetics of a putative chromate reductase from Gluconacetobacter hansenii (Gh-ChrR) was measured and the crystal structure of the protein determined at 2.25 Å resolution. Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions. Kinetic measurements indicate that NADH acts as a substrate inhibitor; catalysis requires chromate binding prior to NADH association. The crystal structure of Gh-ChrR shows the protein is a homotetramer with one bound flavin mononucleotide (FMN) per subunit. A bound anion is visualized proximal to the FMN at the interface between adjacent subunits within a cationic pocket, which is positioned at an optimal distance for hydride transfer. Site-directed substitutions of residues proposed to involve in both NADH and metal anion binding (N85A or R101A) result in 90-95% reductions in enzyme efficiencies for NADH-dependent chromate reduction. In comparison site-directed substitution of a residue (S118A) participating in the coordination of FMN in the active site results in only modest (50%) reductions in catalytic efficiencies, consistent with the presence of a multitude of side chains that position the FMN in the active site. The proposed proximity relationships between metal anion binding site and enzyme cofactors is discussed in terms of rational design principles for the use of enzymes in chromate and uranyl bioremediation.PLoS ONE 01/2012; 7(8):e42432. · 3.73 Impact Factor
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ABSTRACT: Electrophilic compounds such as glyoxals, which are toxic due to their reactive carbonyl group, are generated in vivo through various pathways. In this study, we obtained evidence indicating that the nemRA operon, previously reported to encode a repressor and the N-ethylmaleimide reductase, respectively, is co-transcribed with the 3'-proximal gloA gene encoding glyoxalase I. The operon is not only involved in cytosolic detoxification but is also regulated by electrophiles such as quinones and glyoxals. A gel mobility shift assay revealed that purified NemR repressor bound to DNA was dissociated upon interaction with quinones and glyoxals, while their reduced forms were ineffective. The cysteines of NemR at 21 and 116 were essential in sensing electrophiles in vivo and in vitro. Reversible intermolecular disulphide bonds were observed with a reducing agent as well as with electrophiles. DNA binding affinity reduced by glyoxal was also increased with a reducing agent. The NemA reductase, an FMN-containing enzyme, exhibited catalytic activity toward various electrophiles including quinones, while GloA played a major role in glyoxal detoxification. Therefore, we propose that cells have a cytosolic system consisting of the nemRA-gloA operon for the reduction of electrophiles, especially quinones and glyoxals, to maintain an appropriate intracellular redox balance.Molecular Microbiology 03/2013; · 4.96 Impact Factor