Abstract
Naturally synthesized quinones perform a variety of important cellular functions. Escherichia coli produce both ubiquinone and menaquinone, which are involved in electron transport. However, semiquinone intermediates produced during the one-electron reduction of these compounds, as well as through auto-oxidation of the hydroxyquinone product, generate reactive oxygen species that stress the cell. Here, we present the crystal structure of YgiN, a protein of hitherto unknown function. The three-dimensional fold of YgiN is similar to that of ActVA-Orf6 monooxygenase, which acts on hydroxyquinone substrates. YgiN shares a promoter with "modulator of drug activity B," a protein with activity similar to that of mammalian DT-diaphorase capable of reducing mendione. YgiN was able to reoxidize menadiol, the product of the "modulator of drug activity B" (MdaB) enzymatic reaction. We therefore refer to YgiN as quinol monooxygenase. Modulator of drug activity B is reported to be involved in the protection of cells from reactive oxygen species formed during single electron oxidation and reduction reactions. The enzymatic activities, together with the structural characterization of YgiN, lend evidence to the possible existence of a novel quinone redox cycle in E. coli.
Highlights
Quinones are biologically active molecules that function as lipid electron carriers for the transportation of hydrogen and electrons between the protein complexes of the electron transport chain
It has been demonstrated that the activity of this enzyme protects rat hepatocytes from menadione toxicity by competing with the potentially toxic one-electron reduction pathway employed by the electron transport chain and avoiding the generation of a semiquinone intermediate (6 – 8)
A structural homology search yielded a good alignment with the structure of ActVA-Orf6, a novel monooxygenase from S. coelicolor, with a 1.9 Å root mean square deviation [15] (Fig. 1b)
Summary
Crystallization and Data Collection—Recombinant QuMo (YgiN) was expressed in BL21 (DE3) E. coli cells under the T7 promoter. Co-crystals with menadione were generated by dissolving menadione sodium bisulfite in 50 mM NaH2PO4, pH 8, 300 mM NaCl followed by incubation with purified QuMo protein in a 20:1 ligand-toprotein molar ratio. Data from the native QuMo crystal were collected at the F1 beamline at Cornell High Energy Synchrotron Source (Ithaca, NY) equipped with an ADSC Quantum-4 CCD detector. QuMo-menadione complex data were collected at the X6-A beamline, at the NSLS, using an ADSC Quantum-210 CCD detector. The final model was built using the refined phases by ARP/wARP 6.0 [23] This model contained 103 of 104 residues from the native. The coordinates for the native protein have been deposited in the Protein Data Bank under the accession code 1R6Y. The activity of QuMo was demonstrated by an increase in absorbance at 308 nm corresponding to the oxidation of menadiol to generate menadione
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