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Amino Acid Residues Interacting with Both the Bound Quinone and Coenzyme, Pyrroloquinoline Quinone, in Escherichia coli Membrane-bound Glucose Dehydrogenase

2008; Elsevier BV; Volume: 283; Issue: 32 Linguagem: Inglês

10.1074/jbc.m800911200

ISSN

1083-351X

Autores

Golam Mustafa, Yoshinori Ishikawa, Kazuo Kobayashi, Catharina T. Migita, MD. Elias, Satsuki Nakamura, Seiichi Tagawa, Mamoru Yamada,

Tópico(s)

Metal-Catalyzed Oxygenation Mechanisms

Resumo

The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (eaq-) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of eaq-, although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH. The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (eaq-) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of eaq-, although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH. The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) 2The abbreviations used are: mGDHmembrane-bound glucose dehydrogenasePQQpyrroloquinoline quinoneUQubiquinoneDMN-dodecyl β-d-maltosideMOPS3-(N-morpholino)propanesulfonic acidKPBpotassium phosphate bufferPMSphenazine methosulfateeaq-hydrated electronHPLChigh pressure liquid chromatography. 2The abbreviations used are: mGDHmembrane-bound glucose dehydrogenasePQQpyrroloquinoline quinoneUQubiquinoneDMN-dodecyl β-d-maltosideMOPS3-(N-morpholino)propanesulfonic acidKPBpotassium phosphate bufferPMSphenazine methosulfateeaq-hydrated electronHPLChigh pressure liquid chromatography. belongs to the quinoprotein family with PQQ as a coenzyme (1Duine J.A. Frank J. van Zeeland J.K. FEBS Lett. 1979; 108: 443-446Crossref PubMed Scopus (232) Google Scholar, 2Ameyama M. Matsushita K. Ohno Y. Shinagawa E. Adachi O. FEBS Lett. 1981; 130: 179-183Crossref PubMed Scopus (131) Google Scholar), and it catalyzes d-glucose oxidation to d-gluconate at the periplasmic side to transfer electrons to ubiquinol oxidase via UQ in the respiratory chain (3Van Schie B.J. Hellingwerf K.J. Van Dijken J.P. Elferink M.G.L. Van Dijl J.M. Kuenen J.G. Konings W.L. J. Bacteriol. 1985; 163: 493-499Crossref PubMed Google Scholar, 4Matsushita K. Nonobe M. Shinagawa E. Adachi O. Ameyama M. J. Bacteriol. 1987; 169: 205-209Crossref PubMed Google Scholar, 5Yamada M. Sumi K. Matsushita K. Adachi O. Yamada Y. J. Biol. Chem. 1993; 268: 12812-12817Abstract Full Text PDF PubMed Google Scholar). Topological analysis revealed that mGDH consists of an N-terminal hydrophobic domain with five membrane-spanning segments and a large C-terminal domain residing in the periplasm, which contains PQQ and Ca2+- or Mg2+-binding sites in a superbarrel structure, conserved in quinoproteins (6Cozier G.E. Anthony C. Biochem. J. 1995; 312: 679-685Crossref PubMed Scopus (64) Google Scholar, 7Hommes R.W.J. Postma P.W. Neijssel O.M. Tempest D.W. Dokter P. Duine J.A. FEMS Microbiol. Lett. 1984; 24: 329-333Crossref Scopus (103) Google Scholar, 8Matsushita K. Arents J.C. Bader R. Yamada M. Adachi O. Postma P.W. Microbiology. 1997; 143: 3149-3156Crossref PubMed Scopus (85) Google Scholar). Although its tertiary structure has not been resolved, the arrangement of amino acid residues around PQQ has been modeled on the basis of the crystal structure of the quinoprotein methanol dehydrogenase (6Cozier G.E. Anthony C. Biochem. J. 1995; 312: 679-685Crossref PubMed Scopus (64) Google Scholar) as depicted in Fig. 1. The arrangement has been confirmed by results of several experiments with site-directed amino acid substitutions (9Sode K. Kojima K. Biotechnol. Lett. 1997; 19: 1073-1077Crossref Scopus (28) Google Scholar, 10Yamada M. Inbe H. Tanaka M. Sumi K. Matsushita K. Adachi O. J. Biol. Chem. 1998; 273: 22021-22027Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Elias M.D. Tanaka M. Izu H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2000; 275: 7321-7326Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 12Cozier G.E. Salleh R.A. Anthony C. Biochem. J. 1999; 340: 639-647Crossref PubMed Google Scholar, 13Okuda J. Yoshida H. Kojima K. Himi M. Sode K. J. Biochem. Mol. Biol. & Biophys. 2000; 4: 415-422Google Scholar). The orthoquinone portion of PQQ is a vital part for the catalytic reaction, to which Lys-493 hydrogen-bonds in the model mGDH. Asp-466 is located close to the portion and functions in extracting a proton from glucose. membrane-bound glucose dehydrogenase pyrroloquinoline quinone ubiquinone N-dodecyl β-d-maltoside 3-(N-morpholino)propanesulfonic acid potassium phosphate buffer phenazine methosulfate hydrated electron high pressure liquid chromatography. membrane-bound glucose dehydrogenase pyrroloquinoline quinone ubiquinone N-dodecyl β-d-maltoside 3-(N-morpholino)propanesulfonic acid potassium phosphate buffer phenazine methosulfate hydrated electron high pressure liquid chromatography. Several respiratory components, including primary dehydrogenases, have been found to possess bound UQ in their molecules (14Peter L.J. Anthony C. Biochim. Biophys. Acta. 2003; 1647: 200-205Crossref PubMed Scopus (11) Google Scholar, 15Yankovskaya V. Horsefield R. Tornroth S. Luna-Chavez C. Miyoshi H. Leger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (682) Google Scholar, 16Gong X. Xie T. Yu L. Hesterberg M. Scheide D. Friedrich T. Yu C. J. Biol. Chem. 2003; 278: 25731-25737Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 17Matsushita K. Yakushi T. Toyoma H. Adachi O. Miyoshi H. Tagami E. Sakamoto K. Biochim. Biophys. Acta. 1999; 1409: 154-164Crossref PubMed Scopus (16) Google Scholar, 18Cecchini G. Schröder I. Robert P. Maklashina G. Maklashina E. Biochim. Biophys. Acta. 2002; 1553: 140-157Crossref PubMed Scopus (204) Google Scholar, 19Maklashina E. Hellwig P. Rothery R.A. Kotlyar V. Sher Y. Weiner J.H. Cecchini G. J. Biol. Chem. 2006; 281: 26655-26664Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 20Salerno J.C. Ohnishi T. Biochem. J. 1980; 192: 769-781Crossref PubMed Scopus (69) Google Scholar, 21Yamashita T. Nakamura E. Miyoshi H. Matsuno A. Yagi T. J. Biol. Chem. 2007; 282: 6012-6020Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Sun F. Huo X. Zhai Y. Wang A. Xu J. Su D. Bartlam M. Rao Z. Cell. 2005; 121: 1043-1057Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar), and in some of them, their local structures surrounding UQ have been disclosed. A single bound UQ was found in the crystal structures in succinate dehydrogenase (15Yankovskaya V. Horsefield R. Tornroth S. Luna-Chavez C. Miyoshi H. Leger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (682) Google Scholar) and from studies of mutants and Q-site inhibitors of the subunit of type I NADH dehydrogenase (16Gong X. Xie T. Yu L. Hesterberg M. Scheide D. Friedrich T. Yu C. J. Biol. Chem. 2003; 278: 25731-25737Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), but it is not clear whether a second site exists. The yeast mitochondrial type II NADH dehydrogenase has been demonstrated to have two UQ-binding sites (21Yamashita T. Nakamura E. Miyoshi H. Matsuno A. Yagi T. J. Biol. Chem. 2007; 282: 6012-6020Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), but the tertiary structure still remains to be resolved. The E. coli cytochrome bo ubiquinol oxidase has two UQ-binding sites with high (QH) and with low (QL) affinity for UQ (23Sato-Watanabe M. Mogi T. Miyoshi H. Iwamura H. Matsushita K. Adachi O. Anraku Y. J. Biol. Chem. 1994; 269: 28899-28907Abstract Full Text PDF PubMed Google Scholar, 24Sato-Watanabe M. Mogi T. Ogura T. Kitagawa T. Miyoshi H. Iwamura H. Anraku Y. J. Biol. Chem. 1994; 269: 28908-28912Abstract Full Text PDF PubMed Google Scholar). The crucial amino acid residues interacting with UQ at QH have been identified and shown to be conserved in cytochrome bo or bd ubiquinol oxidases (25Abramson J. Riistama S. Larrson G. Jasaitis A. Svensson-Ek M. Laakkonen L. Puustinen A. Iwata S. Wikström M. Nat. Struct. Biol. 2000; 7: 910-917Crossref PubMed Scopus (358) Google Scholar). The UQ-binding site adopts a binding conformation similar to that of UQ-binding sites (QI and Qo) of the cytochrome bc1 complex in bovine mitochondria (26Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1062) Google Scholar). Amino acid residues interacting with bound UQ in the E. coli cytochrome bd ubiquinol oxidase (27Matsumoto Y. Murai M. Fujita D. Sakamoto K. Miyoshi H. Yoshida M. Mogi T. J. Biol. Chem. 2006; 281: 1905-1912Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and in the E. coli DsbB related to the formation of disulfide bridges in secreted proteins (28Inaba K. Murakami S. Suzuki M. Nakagawa A. Yamashita E. Okada K. Ito K. Cell. 2006; 127: 789-801Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) have also been reported. However, little information on the local structure or physiological function of bound UQ in primary components of respiratory chains has so far been available. Although more than a dozen quinoprotein dehydrogenases have been discovered, little is known about their intramolecular electron transfer pathways. The E. coli mGDH has been demonstrated to have two UQ-binding sites, one (QI) for bound UQ and the other (QII) for bulk UQ (29Elias M.D. Nakamura S. Migita C.T. Miyoshi H. Toyama H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2004; 279: 3078-3083Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), which is near the membrane surface rather than in the hydrophobic interior (30Miyoshi H. Niitome Y. Matsushita K. Yamada M. Iwamura H. Biochim. Biophys. Acta. 1999; 1412: 29-36Crossref PubMed Scopus (13) Google Scholar). In succession to the catalytic reaction, electrons from PQQH2 would be transferred directly to UQ in the QII site or via bound UQ. Our previous study revealed that a eaq- produced by pulse radiolysis caused rapid reduction of a bound UQ, followed by intramolecular electron transfer to PQQ in mGDH. The interdomain electron transfer from UQ to PQQ occurs in both directions, and the distance, 11-13 Å, between PQQ and bound UQ (31Kobayashi K. Mustafa G. Tagawa S. Yamada M. Biochemistry. 2005; 44: 13567-13572Crossref PubMed Scopus (20) Google Scholar) is similar to the two redox centers of copper-containing nitrate reductase (37Kobayashi K. Koppenhöer A. Ferguson S.J. Tagawa S. Biochemistry. 1997; 36: 13611-13616Crossref PubMed Scopus (60) Google Scholar) or heme-heme edge distance of cd1 nitrite reductase (32Fülöp V. Moir J.W.B. Ferguson S.J. Hajdu J. Cell. 1995; 81: 369-377Abstract Full Text PDF PubMed Scopus (251) Google Scholar). However, our knowledge of the local structure of the bound UQ site has so far been limited. The E. coli mGDH is a good model for primary dehydrogenases in terms of occurrence as a single protein and as an apoprotein, which can be easily purified and reconstituted with its coenzyme PQQ (5Yamada M. Sumi K. Matsushita K. Adachi O. Yamada Y. J. Biol. Chem. 1993; 268: 12812-12817Abstract Full Text PDF PubMed Google Scholar, 34Ameyama M. Nonobe M. Hayashi M. Shinagawa E. Matsushita K. Adachi O. Agric. Biol. Chem. 1985; 49: 1227-1231Google Scholar). This model might provide valuable information on the physiological function of bound UQ and on the intramolecular electron transfer. In this study, we identified amino acid residues interacting with bound UQ and proposed their functions in intramolecular events of the enzyme. Clues for this issue have been obtained by previous studies that revealed very close location of the bound UQ to PQQ (31Kobayashi K. Mustafa G. Tagawa S. Yamada M. Biochemistry. 2005; 44: 13567-13572Crossref PubMed Scopus (20) Google Scholar) and the position and functions of Asp-466 and Lys-493 flanking PQQ (11Elias M.D. Tanaka M. Izu H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2000; 275: 7321-7326Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Site-directed mutants of Asp-354, Asp-466, and Lys-493 had significant effects on the characteristics of UQ semiquinone radicals or UQ EPR signals and/or the affinity for UQ. Therefore, these amino acid residues seem to be crucial not only for the catalytic reaction but also for the function of bound UQ. We discuss the possible interaction between the catalytic reaction and the intramolecular electron transfer in mGDH. Materials—UQn was kindly provided by Eizai Co., Ltd. (Japan). All other chemicals were of analytical grade and purchased from commercial sources. Bacterial Strains and Plasmids—E. coli YU423 (Δ(ptsH ptsI crr) galP::Tn10 gcd::cm) (8Matsushita K. Arents J.C. Bader R. Yamada M. Adachi O. Postma P.W. Microbiology. 1997; 143: 3149-3156Crossref PubMed Scopus (85) Google Scholar) and MV1184 (Δ(lac-proAB) ara rpsL thi (φ80 lacZΔM15) Δ(srl-recA) 306::Tn10 (tetr)/F′[traD36 proAB+ lacIq lacZΔM15]) (Takara Shuzo, Japan) strains were used as host strains to express mutant mGDHs and to introduce the mutation of D354N into the mGDH gene (gcd), respectively. Plasmids used were pUCGCD1 bearing the wild-type gcd (10Yamada M. Inbe H. Tanaka M. Sumi K. Matsushita K. Adachi O. J. Biol. Chem. 1998; 273: 22021-22027Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), pUCGCDK493A bearing K493A gcd, pUCGCDK493R bearing K493R gcd, pUCGCDD466E bearing D466E gcd, pUCGCDD466N bearing D466N gcd (11Elias M.D. Tanaka M. Izu H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2000; 275: 7321-7326Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), and pUCGCDD354N bearing D354N gcd. Construction of D354N Mutant—To construct the D354N mGDH mutant, site-specific mutagenesis was carried out using the Mutan™-Super Express Km kit (Takara Shuzo) as described previously (11Elias M.D. Tanaka M. Izu H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2000; 275: 7321-7326Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The mutagenic primer used for the mutant was 5′-CAGTTACCaatAACTTCTC-3′. Purification of Wild-type and Mutant mGDHs—YU423 cells harboring the wild-type pUCGCD1 or harboring one of the pUCGCDs encoding mutant mGDH were grown in LB medium (1% Bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl) containing ampicillin (50 μg/ml) for 12 h at 30 °C under aerobic conditions. Harvesting of cells and preparation of membrane fractions were carried out as described by Yamada et al. (5Yamada M. Sumi K. Matsushita K. Adachi O. Yamada Y. J. Biol. Chem. 1993; 268: 12812-12817Abstract Full Text PDF PubMed Google Scholar). Purification of mGDH from membrane fractions was performed at 4 °C as described previously (29Elias M.D. Nakamura S. Migita C.T. Miyoshi H. Toyama H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2004; 279: 3078-3083Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) with some modifications. In particular, to improve the recovery of UQ-containing mGDH, we avoided using chloride-containing buffer and Triton X-100, and dialysis after the first column chromatography was eliminated. Membrane fractions (∼10 mg/ml of protein) were treated with 10 mm KPB (pH 7.0) containing 0.04% DM and centrifuged 86,000 × g for 90 min, and the resultant membrane fractions were subjected to solubilization for 60 min in the presence of 100 mm KPB (pH 7.0) containing 0.2% DM. The suspension was centrifuged at 86,000 × g for 90 min, and the supernatant obtained was dialyzed against the 10 mm KPB buffer (pH 7.0) without DM. The dialysate was applied onto a DEAE-Toyopearl column (1-ml bed volume/about 10 mg of protein) equilibrated with 10 mm KPB (pH 7.0) containing 0.1% DM. The column was washed with 10-bed volumes of the same buffer and successively with 10-bed volumes of 10 mm KPB (pH 7.0) containing 0.02% DM. The enzyme was eluted by a linear gradient composed of 10-bed volumes of 25 mm KPB buffer (pH 7.0) containing 0.02% DM and 10-bed volumes of 130 mm KPB (pH 7.0) containing 0.02% DM. Active fractions eluted were pooled and directly applied onto a ceramic hydroxyapatite column (1-ml bed volume/about 5 mg of protein) equilibrated with 10 mm KPB (pH 7.0) containing 0.02% DM. The column was washed with 10-bed volumes of 200 mm KPB (pH 7.0) containing 0.02% DM. mGDH was eluted by a linear gradient composed of 10-bed volumes of 200 mm KPB (pH 7.0) containing 0.02% DM and 10-bed volumes of 1 m KPB (pH 7.0) containing 0.02% DM. Active fractions at about 700 mm KPB were pooled and dialyzed against 10 mm KPB (pH 7.0). The active fractions dialyzed were concentrated by a DEAE-Toyopearl column (1-ml bed volume/about 10 mg of protein), in which the enzyme adsorbed was eluted with a small volume of 150 mm KPB (pH 7.0) containing 0.1% DM. These concentrated materials were found to have homogeneity of more than 95%, judging from SDS-7% polyacrylamide gel electrophoresis, and were used as purified mGDHs. Measurement of Protein and Enzyme Activities—Protein content was determined according to the Dulley and Grieve method (35Dulley J.R. Grieve P.A. Anal. Biochem. 1975; 64: 136-141Crossref PubMed Scopus (853) Google Scholar) using bovine serum albumin as a standard. Holoenzyme formation was performed by incubating membrane fractions or purified mGDH in 10 mm MOPS (pH 7.0) containing 30 μm PQQ and 1 mm MgCl2 for 30 min at 25 °C. Using the holoenzyme thus prepared, the following enzyme activities were measured. PMS reductase activity was measured spectrophotometrically (U-2000A, Hitachi) with PMS and 2,4-dichlorophenol indophenol as an electron mediator and acceptor, respectively, as described previously (3Van Schie B.J. Hellingwerf K.J. Van Dijken J.P. Elferink M.G.L. Van Dijl J.M. Kuenen J.G. Konings W.L. J. Bacteriol. 1985; 163: 493-499Crossref PubMed Google Scholar). UQ2 reductase activity was also measured spectrophotometrically in the presence of 0.0025% Tween 20 (11Elias M.D. Tanaka M. Izu H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2000; 275: 7321-7326Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 42Elias M.D. Tanaka M. Sakai M. Toyama H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2001; 276: 48356-48361Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The contents of mGDH protein in membrane fractions were compared by Western blot analysis using an antibody against E. coli mGDH (36Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). CD Analysis—Far-ultraviolet (200-260 nm) circular CD (far-UV CD) spectra were recorded at 25 °C on a J-600 spectropolarimeter (Jasco) with a 1.0-cm cuvette, by which the conformation of purified mutant mGDHs (2.3 μm) in 10 mm KPB (pH 7.0) was compared with that of the wild-type mGDH. Determination of UQ Content in Mutant mGDHs—Three nmol of enzyme was treated with 10 volumes of 100% ethanol in the presence of 3 nmol of UQ6 as an internal standard and incubated for 30 min by mild shaking at 30 °C. The solution was centrifuged at 14,000 rpm for 10 min to remove denatured protein molecules. The supernatant was mixed with 2.5 volumes of n-hexane for 1 min. The upper phase was collected and dried, and the residue was then resolved in 0.2 ml of HPLC solvent (ethanol/methanol/acetonitrile, 4:3:3 v/v). The resolved materials were subjected to reverse-phase HPLC using a Zorbax ODS column (Mitsuitoatsu, Japan) at a flow rate of 0.8 ml/min. The elution was monitored at 278 nm by using an SPD-M6A photodiode array detector (Shimadzu, Japan). UQ from purified mGDH samples was identified by comparison of its migration with those of standard UQs (UQ8 and UQ6), and the content was estimated from the ratio of the peak area to that of the internal standard UQ6. Pulse Radiolysis Analysis of Mutant mGDHs—Pulse radiolysis experiments with purified mutant and wild-type mGDHs were performed on a linear accelerator at the Institute of Scientific and Industrial Research, Osaka University (37Kobayashi K. Koppenhöer A. Ferguson S.J. Tagawa S. Biochemistry. 1997; 36: 13611-13616Crossref PubMed Scopus (60) Google Scholar, 38Suzuki S. Kohzuma T. Deligeer Yamguchi K. Nakamura N. Shidara S. Kobayashi K. Tagawa S. J. Am. Chem. Soc. 1994; 116: 11145-11146Crossref Scopus (79) Google Scholar, 39Kobayashi K. Tagawa S. Daff S. Sagami I. Shimizu T. J. Biol. Chem. 2001; 276: 39864-39871Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 40Kobayashi K. Tagawa S. J. Am. Chem. Soc. 2003; 125: 10213-10218Crossref PubMed Scopus (211) Google Scholar). The pulse width and energy were 8 ns and 27 MeV, respectively. Samples for pulse radiolysis were prepared in 10 mm potassium phosphate (pH 7.4) and 0.1 m of tert-butyl alcohol (for scavenging OH radicals) and subjected to repeated deaeration followed by flushing with argon gas. Each sample was placed in a quartz cell with an optical path length of 1 cm. The temperature of the sample was maintained at 25 °C. The light source for the spectrophotometer was a 200-watt xenon lamp. After passing through an optical path, the transmitted light intensities were analyzed and monitored by a fast spectrophotometric system composed of a Nikon monochromator, a photomultiplier (Hamamatsu Photonics, R-928), and a Unisoku data analyzing system. For time-resolved transient absorption spectral measurement, the monitor light was focused into quartz optical fiber, which transported the electron pulse-induced transmittance changes to a gated spectrometer (Unisoku, TSP-601-02). The concentration of eaq- generated by pulse radiolysis was determined by absorbance change at 650 nm using an extinction coefficient of 14.1 mm-1 cm-1 (41Gordon S. Hart E.J. J. Am. Chem. Soc. 1964; 86: 5343-5344Crossref Scopus (23) Google Scholar) and was adjusted by varying the dose of the electron beam. EPR Spectroscopy—Two hundred μl of concentrated enzyme solution (60-80 μm) in an extra high quality quartz tube (5 mm in outer diameter) was frozen in liquid nitrogen. EPR experiments were carried out on a Bruker E500 spectrophotometer equipped with an hsw10106 resonator and an Oxford E900 cryostat under the same conditions as those described by Elias et al. (29Elias M.D. Nakamura S. Migita C.T. Miyoshi H. Toyama H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2004; 279: 3078-3083Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), except that PQQ was not added for detection of the semiquinone radical of bound UQ. Structural Integrity and Enzyme Activity of Purified Mutant mGDHs—In our previous study, it was shown that Asp-466 and Lys-493, located in close proximity to PQQ, are crucial for a successive process of catalytic reactions from glucose oxidation to UQ reduction. Therefore, we assumed that these amino acid residues interact directly or indirectly with bound UQ, and mutation of these residues appears to impair electron transfer between the bound UQ and PQQ. This assumption was tested with five mutants, D354N, D466E, D466N, K493A, and K493R. Of these, D354N mutant was constructed in this study. Western blot analysis with membrane fractions from cells expressing each mutant mGDH revealed that all mutant mGDHs were expressed at a level similar to that of the wild-type mGDH (11Elias M.D. Tanaka M. Izu H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2000; 275: 7321-7326Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) (data not shown for D354N). In the purification process with two column chromatographies, elution patterns of these mutant mGDHs were found to be almost the same as that of the wild-type mGDH. We further checked effects of mutations on the protein structure by CD analysis with the purified mutant proteins. The results showed that there was almost no difference from that of the wild-type mGDH in the five mutants (Fig. 2 and data not shown), suggesting that these mutants retained a conformation almost unaltered compared with that of the wild-type mGDH. PMS reductase activity reflecting glucose dehydrogenase activity of purified mGDH mutants was then compared with that of the purified wild-type mGDH (Table 1). As reported previously (10Yamada M. Inbe H. Tanaka M. Sumi K. Matsushita K. Adachi O. J. Biol. Chem. 1998; 273: 22021-22027Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Elias M.D. Tanaka M. Izu H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2000; 275: 7321-7326Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), all mutants except for D354N showed a significantly low level of activity, being less than 0.04% that of the wild-type mGDH, and D354N showed a slightly higher level of activity (about 2.0%) than those of other mutants. Consistent with the level of dehydrogenase activity, UQ2 reductase activities of these mutants were remarkably reduced. No significant increase both in reductase activities was observed under saturation conditions of electron acceptors in any mutant or wild-type mGDHs. These data suggest that the mutants examined maintain an intrinsic structure of the protein but lose almost all of catalytic activity, which agrees with results of previous studies (10Yamada M. Inbe H. Tanaka M. Sumi K. Matsushita K. Adachi O. J. Biol. Chem. 1998; 273: 22021-22027Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Elias M.D. Tanaka M. Izu H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2000; 275: 7321-7326Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar).TABLE 1PMS and UQ2 reductase activities and bound UQ content of purified mutant and wild-type mGDHsPurified enzymePMS reductase activityUQ2 reductase activityBound UQ contentunits/mgunits/mgmol/mol of mGDHWild type540550.9 ± 0.02K493A0.2NDaND indicates not detected.0.1 ± 0.02K493R0.08ND0.9 ± 0.02D466ENDND0.1 ± 0.02D466N0.04ND0.2 ± 0.03D354N9.20.30.5 ± 0.02a ND indicates not detected. Open table in a new tab Effect of Mutations on UQ Content in Purified mGDHs—If Asp-354, Asp-466, or Lys-493 occurs close to or directly interacts with bound UQ, it is possible that the corresponding mGDH mutants alter the affinity for UQ. To examine this possibility, the content of UQ in each purified mutant mGDH was estimated by reverse-phase HPLC after elution of bound UQ from purified enzymes (Table 1). A compound corresponding to the position of UQ8 was detected at 19 min of retention time, with an absorption spectrum having a peak at 278 nm as reported previously (29Elias M.D. Nakamura S. Migita C.T. Miyoshi H. Toyama H. Matsushita K. Adachi O. Yamada M. J. Biol. Chem. 2004; 279: 3078-3083Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). As a result, about 1

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