Structural and Biochemical Evidence for an Enzymatic Quinone Redox Cycle in Escherichia coli

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. 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. 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. Both ubiquinone and menaquinone are synthesized by Escherichia coli for this purpose. However, although these compounds are essential for normal electron transport, it has been demonstrated that such quinonoids are also capable of diverting electron flow from the respiratory chain, and in doing, so cause increased intracellular production of superoxide radicals and hydrogen peroxide (1Hassan H.M. Fridovich I. Arch. Biochem. Biophys. 1979; 196: 385-395Crossref PubMed Scopus (445) Google Scholar). Benzoquinones and napthoquinones, such as menaquinone, are highly electrophilic and exhibit substantial thiol reactivity that results in their rapid reduction to semiquinone intermediates. This high redox potential is responsible for quinone cytotoxicity (2O'Brien P.J. Chem. Biol. Interact. 1991; 80: 1-41Crossref PubMed Scopus (1021) Google Scholar), which causes the oxidation of cellular macromolecules (3Bolton J.L. Trush M.A. Penning T.M. Dryhurst G. Monks T.J. Chem. Res. Toxicol. 2000; 13: 135-160Crossref PubMed Scopus (1403) Google Scholar). It is therefore extremely important that the cell maintains sufficient quinone levels for respiration while minimizing their toxicity. In addition to potential damage from endogenously synthesized quinones, organisms are also at risk of from environmental quinones, such as from the natural antibiotic compounds synthesized by Gram-positive bacteria. The ubiquitous distribution of quinonoids thus requires organisms to possess a specific defense mechanism. The metabolism and toxicity of menadione 1The abbreviations and trivial names used are: menadione, 2-methyl-1,4-naphthoquinone; MdaB, modulator of drug activity B; QuMo, quinol monooxygenase; DT-diaphorase, NAD(P)H:oxidoreductase. (2-methyl-1,4-naphthoquinone) have been extensively studied in mammalian cells (4Brunmark A. Cadenas E. Lind C. Segura-Aguilar J. Ernster L. Free Radic. Biol. Med. 1987; 3: 181-188Crossref PubMed Scopus (53) Google Scholar, 5Iyanagi T. Yamazaki I. Biochim. Biophys. Acta. 1970; 216: 282-294Crossref PubMed Scopus (309) Google Scholar). Menadione is an analogue of menaquinone that differs only in the lack of an extended alkyl chain. DT-diaphorase, an NAD(P)H:oxidoreductase, is believed to reduce a variety of quinone substrates by means of a two-electron reduction mechanism. 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 (6Lind C. Hochstein P. Ernster L. Arch. Biochem. Biophys. 1982; 216: 178-185Crossref PubMed Scopus (502) Google Scholar, 7Lind C. Cadenas E. Hochstein P. Ernster L. Methods Enzymol. 1990; 186: 287-301Crossref PubMed Scopus (288) Google Scholar, 8Thor H. Smith M.T. Hartzell P. Bellomo G. Jewell S.A. Orrenius S. J. Biol. Chem. 1982; 257: 12419-12425Abstract Full Text PDF PubMed Google Scholar). The protein “modulator of drug activity B” (MdaB) was first identified on the basis of the protection it provided from the toxic effects of DMP840, adriamycin, and etoposide when overexpressed in E. coli (9Chatterjee P.K. Sternberg N.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8950-8954Crossref PubMed Scopus (34) Google Scholar). It was later confirmed through protein sequencing that this protein corresponded to a DT-diaphorase-like enzyme previously purified from E. coli that demonstrated reductase activity toward menadione (10Hayashi M. Ohzeki H. Shimada H. Unemoto T. Biochim. Biophys. Acta. 1996; 1273: 165-170Crossref PubMed Scopus (22) Google Scholar). Also, MdaB was reported to be up-regulated more than 20-fold in the cytoplasmic fraction of E. coli in response to 0.2-0.3 mm menadione (11Hayashi M. Hasegawa K. Oguni Y. Unemoto T. Biochim. Biophys. Acta. 1990; 1035: 230-236Crossref PubMed Scopus (26) Google Scholar). Although early work suggested FMN and NADH dependence, later studies found MdaB to be FAD- and NADPH-dependent (10Hayashi M. Ohzeki H. Shimada H. Unemoto T. Biochim. Biophys. Acta. 1996; 1273: 165-170Crossref PubMed Scopus (22) Google Scholar). Confusion as to the co-factor identities still remains. It has been suggested that induction of this enzyme may be an adaptive cellular response to minimize the toxicity of menaquinone and other quinone compounds. Recent studies of the MdaB homologue from Helicobacter pylori have demonstrated that the growth of mdab-deficient mutants is significantly inhibited by 10% oxygen environments and that these bacteria are diminished in their ability to colonize the stomachs of mice when compared with wild-type H. pylori (12Wang G. Maier R.J. Infect. Immun. 2004; 72: 1391-1396Crossref PubMed Scopus (101) Google Scholar). Although this work suggests that MdaB is particularly important for the protection of cells against oxidative stress, the quinol products of the reduction reaction are also capable of generating reactive oxygen species through auto-oxidative processes. Therefore, a detoxification mechanism involving MdaB must also include a mechanism for detoxifying the quinol reduction product. We have expressed a hypothetical open reading frame encoding the putative protein YgiN from E. coli K-12 and determined its three-dimensional crystal structure. Prior to structural determination, the function of this protein was unknown as no sequence conservation with proteins of known function could be detected. Using the RegulonDB data base (www.cifn.unam.mx/Computational_Genomics/regulondb/), a search of the E. coli genome positioned the b3029/YgiN gene immediately down-stream of the modulator of drug activity B (MdaB) gene and predicted that both proteins would be co-regulated by a single promoter (13Salgado H. Gama-Castro S. Martinez-Antonio A. Diaz-Peredo E. Sanchez-Solano F. Peralta-Gil M. Garcia-Alonso D. Jimenez-Jacinto V. Santos-Zavaleta A. Bonavides-Martinez C. Collado-Vides J. Nucleic Acids Res. 2004; 32: D303-D306Crossref PubMed Google Scholar). In this report, we show that the three-dimensional fold of YgiN aligns well with that of ActVA-Orf6, a novel quinone monooxygenase from Streptomyces coelicolor thought to function via a two-election oxidation of quinol substrates in antibiotic biosynthesis pathways (14Kendrew S.G. Hopwood D.A. Marsh E.N. J. Bacteriol. 1997; 179: 4305-4310Crossref PubMed Google Scholar, 15Sciara G. Kendrew S.G. Miele A.E. Marsh N.G. Federici L. Malatesta F. Schimperna G. Savino C. Vallone B. EMBO J. 2003; 22: 205-215Crossref PubMed Scopus (141) Google Scholar). We have also determined the structure of YgiN complexed with menadione. Further, we provide in vitro evidence that MdaB and YgiN form an enzymatic quinone reduction-oxidation cycle. We shall therefore refer to YgiN as quinol monooxygenase (QuMo). It is possible that such a redox cycle allows the cell to maintain a stable pool of quinones for electron transport while attenuating the potential toxicity of both quinone and quinol species. Crystallization and Data Collection—Recombinant QuMo (YgiN) was expressed in BL21 (DE3) E. coli cells under the T7 promoter. The expression construct contained a C-terminal hexa-histidine tag that permitted facile purification using batch elution over nickel-nitrilotriacetic acid resin (Qiagen). The final yield was 380 mg of pure protein/liter E. coli culture with no additional purification steps. All reagents for protein expression and purification were purchased from BIOSHOP Canada. All crystallization reagents were purchased from Hampton Research. The optimal crystallization conditions for the native protein were 1.5 m ammonium sulfate, 100 mm sodium citrate, pH 5.6, 0.2 m potassium/sodium tartrate, at 298 K using hanging drop vapor diffusion and a protein concentration of 15 mg/ml. QuMo protein was also expressed as a selenomethionine derivative in DL41 (DE3) E. coli in LE Master medium (16Hendrickson W.A. Horton J.R. LeMaster D.M. EMBO J. 1990; 9: 1665-1672Crossref PubMed Scopus (1008) Google Scholar) and purified as per native QuMo protein. The crystallization conditions for the selenomethionine protein were unaltered; however the protein concentration was decreased to 2.5 mg/ml, and the volume ratio of protein to reservoir solution was modified from 2:2 to 5:2. 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-to-protein molar ratio. The protein-ligand complex was then crystallized in 1.0 m ammonium sulfate, 0.1 m sodium citrate, pH 5.6, and 0.1 m potassium/sodium tartrate, at 298 K. All data were collected at cryogenic temperature. 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. Single anomalous dispersion data were collected on the X9-A beamline at the National Synchrotron Light Source National NY) during the using a CCD detector. complex data were collected at the also at the using an ADSC CCD detector. Data were using and Methods Enzymol. 1997; PubMed Scopus Google Scholar). The is with cell a for the native The are to the native and QuMo crystal structure was determined by the single anomalous dispersion using a selenomethionine The for of were determined using and were generated using at I. Biol. 9: PubMed Scopus Google Scholar, Methods Enzymol. PubMed Scopus Google Scholar). this the was and of to and involving the native data were using J. Biol. PubMed Scopus Google Scholar, Biol. 2000; PubMed Scopus Google Scholar, Biol. 2003; PubMed Scopus Google Scholar). The were as and the was using from the results with by A. Biol. PubMed Scopus Google Scholar) using Biol. 1997; Scopus Google Scholar). The final was using the by A. Biol. PubMed Scopus Google Scholar). This contained of from the native although no was for the C-terminal the purification are molecules in the final The for the native protein have been in the Data under the were using the at to search the of data base E. K. Biol. 2004; PubMed Scopus Google Scholar). The complex structure was in using the QuMo native structure P. J. M. R.J. T. Biol. PubMed Scopus Google Scholar). was in the active and a menadione was molecules were and the structure was using Biol. 1997; Scopus Google Scholar). The have been in the Data under the of MdaB and menadione reductase activity was at in a reaction 50 mm NaH2PO4, pH 0.2 mm 50 menadione, and enzyme in a volume of 1.0 The activity was by the in at from the of menadione to QuMo activity was by the addition of this enzyme in a molar ratio with MdaB to the reaction menadione been by The activity of QuMo was demonstrated by an in at to the oxidation of to generate menadione. of QuMo It a three-dimensional structure of QuMo from E. coli K-12 was determined using the single anomalous dispersion and to The was to and and with of in the of the and the in The fold is of the of the fold and a Full Text PDF PubMed Scopus Google Scholar) structural search a with the structure of ActVA-Orf6, a novel monooxygenase from S. with a G. Kendrew S.G. Miele A.E. Marsh N.G. Federici L. Malatesta F. Schimperna G. Savino C. Vallone B. EMBO J. 2003; 22: 205-215Crossref PubMed Scopus (141) Google Scholar) Both that form an in which is positioned between and is in sequence of QuMo with of the monooxygenase However, is both and for the found in of this which is in QuMo data and to cell 32: a in a of QuMo is formed with a from a through an that has a of QuMo also a in solution The between the of QuMo a and ActVA-Orf6 also a in which the of and a hydrogen between and these are present in in the ActVA-Orf6 the QuMo a in which the extended between and of to the of the other by to In ActVA-Orf6, this is formed by an extended of the as the extended is The to the of the of the active and to be important for particularly in the of from and of Although the of of in the the of QuMo is to that of ActVA-Orf6 The to the active of ActVA-Orf6 a of and The active of QuMo, is and sequence to the of the protein is in ActVA-Orf6, it is in positioned between and is also between the proteins and to the of In to previously identified QuMo the is by of ActVA-Orf6, is by of this to the of the a between and of ActVA-Orf6 is Although from ActVA-Orf6 is in QuMo, and in ActVA-Orf6 are by and in the of the of that the is highly and results are with the that this is important in the of the of The of this are that substrates. It is therefore that structural are involved in the of the active at the of the results in the of over a Although the of the ActVA-Orf6 is by the the of QuMo is involved in the of the active the of and the of the QuMo active The studies of ActVA-Orf6 have that the enzyme a for (14Kendrew S.G. Hopwood D.A. Marsh E.N. J. Bacteriol. 1997; 179: 4305-4310Crossref PubMed Google Scholar, 15Sciara G. Kendrew S.G. Miele A.E. Marsh N.G. Federici L. Malatesta F. Schimperna G. Savino C. Vallone B. EMBO J. 2003; 22: 205-215Crossref PubMed Scopus (141) Google Scholar, S.G. Federici L. Savino C. Miele A. Marsh E.N. Vallone B. Biol. 2000; PubMed Scopus Google Scholar). This is also the for QuMo and for other of this is no that could be to a a of that could as a The native structure of ActVA-Orf6 suggested that may be important for of the hydroxyquinone and to the and hanging from is the only of these to be in the with and may for the of substrates. The ability of QuMo to compounds was confirmed through of QuMo with menadione, a natural reaction product. The structure was to with menaquinone in the active molecules were also positioned in the active to menadione in the via hydrogen The oxygen in the of menadione is to a which in with the chain of The between the first and the to the oxygen of The of menadione the of the alkyl of the similar to the of of the active structure than the natural product G. Kendrew S.G. Miele A.E. Marsh N.G. Federici L. Malatesta F. Schimperna G. Savino C. Vallone B. EMBO J. 2003; 22: 205-215Crossref PubMed Scopus (141) Google Scholar). QuMo in with MdaB to a with an This was by the of the purified as well as a with a at was only with NADH no activity was when was This the which suggested that this DT-diaphorase-like enzyme in E. coli was (11Hayashi M. Hasegawa K. Oguni Y. Unemoto T. Biochim. Biophys. Acta. 1990; 1035: 230-236Crossref PubMed Scopus (26) Google Scholar). the basis of this also suggests that MdaB is MdaB was able to reduce 50 of menadione in 100 as by the of menadione This is in with previously reported data for the of menadione by a DT-diaphorase-like enzyme purified from E. coli when NADH was provided for reaction (11Hayashi M. Hasegawa K. Oguni Y. Unemoto T. Biochim. Biophys. Acta. 1990; 1035: 230-236Crossref PubMed Scopus (26) Google Scholar). The oxidation of to menadione was in the and of QuMo at a final concentration of was to the MdaB reaction menadione been to in the is in the of QuMo reduction of menadione was in MdaB NADH was no auto-oxidation of in the of QuMo over this that QuMo is responsible for the oxidation of to menadione. reaction was inhibited by the addition of QuMo the gene product, was for as its sequence with proteins of known function The QuMo crystal structure was to The protein fold well with that of the monooxygenase ActVA-Orf6 from S. coelicolor with an of only sequence was the structural of the of which is of this and thought to an important in (14Kendrew S.G. Hopwood D.A. Marsh E.N. J. Bacteriol. 1997; 179: 4305-4310Crossref PubMed Google Scholar, 15Sciara G. Kendrew S.G. Miele A.E. Marsh N.G. Federici L. Malatesta F. Schimperna G. Savino C. Vallone B. EMBO J. 2003; 22: 205-215Crossref PubMed Scopus (141) Google Scholar, S.G. Federici L. Savino C. Miele A. Marsh E.N. Vallone B. Biol. 2000; PubMed Scopus Google Scholar). The of in both QuMo and ActVA-Orf6 the of sequence on the conservation of protein this of novel has been to pathways responsible for the of compounds, the antibiotic pathways in Gram-positive bacteria. ActVA-Orf6 is such an as a enzyme that to a with The existence of of the novel monooxygenase in which are known to suggests a hitherto cellular function for of this and also that the is than The of sequence and lack of conservation of active together with a of structural to Gram-positive bacteria has to structural to its Gram-positive QuMo to other for this a protein mechanism in which the enzyme a and electron from the to yield a form of the which then with has been I. Y. Chem. 1997; PubMed Scopus Google Scholar, B. 32: PubMed Scopus Google Scholar). This a enzyme species. The found in QuMo is the as an important in the reaction. The of menadione in the active is with the of to and product The alkyl in of menadione of the and to be important for The of the allows the of the chain, which to in in menaquinone This be to the ability of to such as G. Kendrew S.G. Miele A.E. Marsh N.G. Federici L. Malatesta F. Schimperna G. Savino C. Vallone B. EMBO J. 2003; 22: 205-215Crossref PubMed Scopus (141) Google Scholar). the structure of ActVA-Orf6, a enzymatic mechanism on the auto-oxidation of was that the is essential for the specific of the from through a hydrogen that the in the active with the as a hydrogen G. Kendrew S.G. Miele A.E. Marsh N.G. Federici L. Malatesta F. Schimperna G. Savino C. Vallone B. EMBO J. 2003; 22: 205-215Crossref PubMed Scopus (141) Google Scholar). This hydrogen the of a of hydrogen to the and the of the to the of a In to ActVA-Orf6, QuMo the and that are believed to be involved in the mechanism of The of with still the of the the of the must be by other in the active and and may also be in to be involved in may a in between the The in the active are with the in between these It has been suggested that the to the active of ActVA-Orf6 substrates G. Kendrew S.G. Miele A.E. Marsh N.G. Federici L. Malatesta F. Schimperna G. Savino C. Vallone B. EMBO J. 2003; 22: 205-215Crossref PubMed Scopus (141) Google Scholar). In QuMo possess a in the active This suggests that the mechanism of QuMo is more specific than as the enzyme must the that during of substrates in the QuMo is of an antibiotic the of E. coli was for as to a possible natural of Both QuMo and MdaB are highly of both proteins be found in of bacteria and also the and only are the proteins their as The of QuMo and MdaB on the promoter that their are an by the that both on substrates. work to MdaB, menadione was identified as a possible to its structural to substrates of the previously identified and the known ability of MdaB to reduce menadione, an analogue of We have been able to the menadione reductase activity previously reported for MdaB and have it to be In we have demonstrated the ability of QuMo to menadione from in QuMo is able to of the of menadione, at an in vitro ability of QuMo to the reaction product of MdaB and form an enzymatic redox cycle. This work is the first to the existence of the quinone redox cycle in E. as well as to that the function of MdaB may be to the activity of protein. studies have demonstrated that MdaB expression is up-regulated more than 20-fold in response to menadione (11Hayashi M. Hasegawa K. Oguni Y. Unemoto T. Biochim. Biophys. Acta. 1990; 1035: 230-236Crossref PubMed Scopus (26) Google Scholar), QuMo expression to be The expression of QuMo in E. coli K-12 has been reported previously I. PubMed Google Scholar). The response of the promoter of QuMo and MdaB to menadione to the reported expression of these proteins under normal cellular conditions suggests that these proteins have a in the potentially in the protection of cells from oxidative The of an enzymatic redox cycle may be it is important for E. coli to maintain an intracellular pool of menadione, which is employed under growth as well as This would a from to respiration under oxygen environmental to the and of quinone compounds, it would be for cells to maintain such the of reactive oxygen species. redox such as the formed by QuMo and MdaB, the of these toxic by that the of a semiquinone intermediate This is by the of reaction to by in the a of single electron reduction which that both proteins through two-electron quinones are in and are by of the these quinones are also able to generate reactive oxygen species. the cycle would also as a quinone to the for these of proteins (12Wang G. Maier R.J. Infect. Immun. 2004; 72: 1391-1396Crossref PubMed Scopus (101) Google Scholar, S.G. Hopwood D.A. Marsh E.N. J. Bacteriol. 1997; 179: 4305-4310Crossref PubMed Google Scholar). In we have the structure of the first of the novel monooxygenase involved in the biosynthesis of compounds. This that these proteins may be more than and suggests that have been identified of the sequence between these and more The structure of QuMo maintains the similar to ActVA-Orf6, sequence However, in the of active as well as in in the a in between the we have provided evidence that the QuMo protein may function with MdaB to form a quinone redox cycle in E. coli that the of species that a substantial to the cell. This work has to only a for the protein also to the of a enzyme with to the of a possible novel quinone redox cycle in E. coli. We P. as well as the of the at the in the of and for and and for the reading of the The of YgiN by M. is We also to M. and A. for the of the MdaB expression construct and their with

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