Crystal Structure of a New Type of NADPH-Dependent Quinone Oxidoreductase (QOR2) from Escherichia coli
Laboratory of Biophysics, School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea. Journal of Molecular Biology
(Impact Factor: 4.33).
06/2008; 379(2):372-84. DOI: 10.1016/j.jmb.2008.04.003
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.
Available from: Changhan Lee
- "Escherichia coli possesses several NADH/NADPHdependent quinone oxidoreductases (Fig. 1), including tryptophan repressor-binding protein WrbA, modulator of drug activity (MdaB), quinone oxidoreductase QoR, chromate reductase YieF and quinone oxidoreductase YtfG (Thorn et al., 1995; Ackerley et al., 2004; Adams and Jia, 2005; Patridge and Ferry, 2006; Kim et al., 2008). They share a structural similarity, each containing a flavin cofactor at the active site, with broad substrate specificities . "
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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; 88(2). DOI:10.1111/mmi.12192 · 4.42 Impact Factor
Available from: Sergio Porté
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ABSTRACT: Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of zeta-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in zeta-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis.
Journal of Biological Chemistry 05/2009; 284(25):17194-205. DOI:10.1074/jbc.M109.001800 · 4.57 Impact Factor
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ABSTRACT: Corynebacterium glutamicum cgR_1435 (cg1552) encodes a protein of the DUF24 protein family, which is a novel family of transcriptional regulators. CgR1435 (QorR) is a negative regulator of cgR_1436 (qor2), which is located upstream of cgR_1435 (qorR) in the opposite orientation, and its structural gene. QorR binds to the intergenic region between qor2 and qorR to repress their expression, which is induced by the thiol-specific oxidant diamide. The DNA-binding activity of QorR is impaired by oxidants such as diamide, H(2)O(2), and cumene hydroperoxide in vitro, and its lone cysteine residue (Cys-17) is essential for redox-responsive regulation of QorR activity both in vivo and in vitro. Moreover, a disruptant of qor2, which is a homologue of the ytfG gene of Escherichia coli encoding quinone oxidoreductase, shows increased sensitivity to diamide. It is concluded that the redox-sensing transcriptional regulator QorR is involved in disulfide stress response of C. glutamicum by regulating qor2 expression.
Journal of Biological Chemistry 05/2009; 284(25):16736-42. DOI:10.1074/jbc.M109.009027 · 4.57 Impact Factor
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