Mutant Isolation of the Escherichia coli Quinoprotein Glucose Dehydrogenase and Analysis of Crucial Residues Asp-730 and His-775 for Its Function

Abstract

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli were obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone. Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli were obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone. Quinoprotein GDH 1The abbreviations used are: GDHglucose dehydrogenaseMDHmethanol dehydrogenaseADHquinohaemoprotein-cytochrome c alcohol dehydrogenaseQ-1ubiquinone-1Q-8ubiquinone-8PCRpolymerase chain reactionPQQpyrroloquinoline quinonePMSphenazine methosulfate. bound to the inner membrane in Escherichia coli functions in a direct oxidation of d-glucose to d-gluconate and concomitantly transferring the electrons to cytochrome oxidase through ubiquinone in the respiratory chain (1Matsushita K. Nonobe M. Shinagawa E. Adachi O. Ameyama M. J. Bacteriol. 1987; 169: 205-209Crossref PubMed Google Scholar, 2Van Schie B.J. Hellingwerf K.J. van Dijken J.P. Elferink M.G.L. van Dijl J.M. Kuenen J.G. Konings W.N. J. Bacteriol. 1985; 163: 493-499Crossref PubMed Google Scholar). Previous reports have indicated that GDH possesses the binding sites for PQQ (3Duine J.A. Frank J. van Zeeland J.K. FEBS Lett. 1979; 108: 443-446Crossref PubMed Scopus (232) Google Scholar, 4Ameyama M. Matsushita K. Ohno Y. Shinagawa E. Adachi O. FEBS Lett. 1981; 130: 179-183Crossref PubMed Scopus (131) Google Scholar), metal ions such as Mg2+ or Ca2+ (5Hommes 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, 6Matsushita K. Arents J.C. Bader R. Yamada M. Adachi O. Postma P.W. Microbiology. 1997; 143: 3149-3156Crossref PubMed Scopus (85) Google Scholar), and ubiquinone (1Matsushita K. Nonobe M. Shinagawa E. Adachi O. Ameyama M. J. Bacteriol. 1987; 169: 205-209Crossref PubMed Google Scholar, 8Matsushita K. Shinagawa E. Adachi O. Ameyama M. J. Biochem. ( Tokyo ). 1989; 105: 633-637Crossref PubMed Scopus (47) Google Scholar, 9Matsushita K. Shinagawa E. Adachi O. Ameyama M. Biochemistry. 1989; 28: 6276-6280Crossref PubMed Scopus (65) Google Scholar) as well as substrate glucose. The enzyme fromE. coli occurs as the apoenzyme (5Hommes 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) because the organism is unable to produce PQQ (6Matsushita K. Arents J.C. Bader R. Yamada M. Adachi O. Postma P.W. Microbiology. 1997; 143: 3149-3156Crossref PubMed Scopus (85) Google Scholar), but is readily reconstituted by incubation with PQQ and the metal ions (7Ameyama M. Nonobe M. Hayashi M. Shinagawa E. Matsushita K. Adachi O. Agric. Biol. Chem. 1985; 49: 1227-1231Google Scholar). To elucidate the functional structure, topological analysis has been performed (10Yamada M. Sumi K. Matsushita K. Adachi O. Yamada Y. J. Biol. Chem. 1993; 268: 12812-12817Abstract Full Text PDF PubMed Google Scholar) which revealed that GDH possesses five membrane-spanning segments at the N-terminal one-sixth portion, and that the remaining C-terminal five-sixth portion occurs at the periplasmic side of the membrane. The large C-terminal portion is assumed to have the catalytic domain including the PQQ-binding site. The binding site of ubiquinone in GDH has been proposed to be at the N-terminal hydrophobic domain of the protein (11Friedrich T. Strohdeicher M. Hofhaus G. Preis D. Sahm H. Weiss H. FEBS Lett. 1990; 265: 37-40Crossref PubMed Scopus (57) Google Scholar). The ubiquinone-binding site has been indicated to be at the region close to the periplasmic side by using reconstituted proteoliposomes (10Yamada M. Sumi K. Matsushita K. Adachi O. Yamada Y. J. Biol. Chem. 1993; 268: 12812-12817Abstract Full Text PDF PubMed Google Scholar), where no membrane potential is generated by the electron transfer from glucose to ubiquinone in the dehydrogenase. glucose dehydrogenase methanol dehydrogenase quinohaemoprotein-cytochrome c alcohol dehydrogenase ubiquinone-1 ubiquinone-8 polymerase chain reaction pyrroloquinoline quinone phenazine methosulfate. Molecular genetic and biochemical analyses of the several quinoprotein dehydrogenases including glucose, methanol, and alcohol dehydrogenases have been performed, and their primary sequences are available (12Harms N. de Vries G.E. Maurer K. Hoogendijk J. Stouthamer A.H. J. Bacteriol. 1987; 169: 3969-3975Crossref PubMed Google Scholar, 13Machlin S.M. Hanson R.S. J. Bacteriol. 1988; 170: 4739-4747Crossref PubMed Google Scholar, 14Cleton-Jansen A.-M. Goosen N. Odle G. Van de Putte P. Nucleic Acids Res. 1988; 16: 6228Crossref PubMed Scopus (35) Google Scholar, 15Cleton-Jansen A.-M. Goosen N. Vink K. van de Putte P. Mol. Gen. Genet. 1989; 217: 430-436Crossref PubMed Scopus (67) Google Scholar, 16Inoue T. Sunagawa M. Mori A. Imai C. Fukuda M. Tagaki M. Yano K. J. Bacteriol. 1989; 171: 3115-3122Crossref PubMed Google Scholar, 17Cleton-Jansen A.-M. Goosen N. Fayet O. van de Putte P. J. Bacteriol. 1990; 172: 6308-6315Crossref PubMed Google Scholar, 18Cleton-Jansen A.-M. Dekker S. van de Putte P. Goosen N. Mol. Gen. Genet. 1991; 229: 206-212Crossref PubMed Scopus (43) Google Scholar, 19Yamada M. Asaoka S. Saier Jr., M.H. Yamada Y. J. Bacteriol. 1993; 175: 568-571Crossref PubMed Google Scholar). Their functional structures appear to be different from each other. Unlike the monomeric and membrane-bound GDH of E. coli (1Matsushita K. Nonobe M. Shinagawa E. Adachi O. Ameyama M. J. Bacteriol. 1987; 169: 205-209Crossref PubMed Google Scholar), Acinetobacter calcoaceticus, orGluconobacter oxydans (14Cleton-Jansen A.-M. Goosen N. Odle G. Van de Putte P. Nucleic Acids Res. 1988; 16: 6228Crossref PubMed Scopus (35) Google Scholar, 17Cleton-Jansen A.-M. Goosen N. Fayet O. van de Putte P. J. Bacteriol. 1990; 172: 6308-6315Crossref PubMed Google Scholar, 18Cleton-Jansen A.-M. Dekker S. van de Putte P. Goosen N. Mol. Gen. Genet. 1991; 229: 206-212Crossref PubMed Scopus (43) Google Scholar, 19Yamada M. Asaoka S. Saier Jr., M.H. Yamada Y. J. Bacteriol. 1993; 175: 568-571Crossref PubMed Google Scholar, 20Anthony C. Int. J. Biochem. 1992; 24: 29-39Crossref PubMed Scopus (50) Google Scholar), the soluble MDH ofMethylobacterium organophilum XX or Paracoccus denitrificans acts as a tetramer, α2β2, to transfer electrons to cytochrome c (13Machlin S.M. Hanson R.S. J. Bacteriol. 1988; 170: 4739-4747Crossref PubMed Google Scholar, 21Anthony C. Chan H.T.C. Cox J.M. Richardson I.W. Murrell J.C. Kelly D.P. Microbial Growth on C1 Compounds. Intercept Ltd., Andover, United Kingdom1993: 221-233Google Scholar, 22Van Spanning R.J.M. Wansell C.W. De Boer T. Hazelaar M.J. Anazawa H. Harms N. Oltmann L.F. Stouthamer A.H. J. Bacteriol. 1991; 173: 6948-6961Crossref PubMed Google Scholar). Whereas, membrane-bound ADH ofAcetobacter aceti occurs as a trimer, αβγ, to transfer electrons via intramolecular heme c moieties to ubiquinone (23Matsushita K. Yakushi T. Toyama H. Shinagawa E. Adachi O. J. Biol. Chem. 1996; 271: 4850-4857Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Alignment of the PQQ-binding protein or subunit in these dehydrogenases revealed that they have three homologous regions including the highly conserved region consisting of about 70 amino acid sequences at their C termini (20Anthony C. Int. J. Biochem. 1992; 24: 29-39Crossref PubMed Scopus (50) Google Scholar), which had been predicted as a common PQQ-binding site (16Inoue T. Sunagawa M. Mori A. Imai C. Fukuda M. Tagaki M. Yano K. J. Bacteriol. 1989; 171: 3115-3122Crossref PubMed Google Scholar, 17Cleton-Jansen A.-M. Goosen N. Fayet O. van de Putte P. J. Bacteriol. 1990; 172: 6308-6315Crossref PubMed Google Scholar). Therefore, the structure of the catalytic proteins of quinoprotein dehydrogenases may be partially similar to each other, but they transfer electrons to different components. Recently, three-dimensional structures of MDHs from three different sources of Methylophilus methylotrophus, Methylophilus W3A1, and Methylobacterium extorquens AM1 (24Xia Z.-X. Dai W.-W. Xiong J.-P. Hao Z.-P. Davidson V.L. White S. Mathews F.S. J. Biol. Chem. 1992; 267: 22289-22297Abstract Full Text PDF PubMed Google Scholar, 25White S. Boyd G. Mathews F.S. Xia Z.-X. Dai W.-W. Zhang Y.-F. Davidson V.L. Biochemistry. 1993; 32: 12955-12958Crossref PubMed Scopus (80) Google Scholar, 26Blake C.C.F. Ghosh M. Harlos K. Avezoux A. Anthony C. Nat. Struct. Biol. 1994; 1: 102-105Crossref PubMed Scopus (89) Google Scholar) have been determined, revealing that the amino acid with PQQ and Ca2+ are in the which a structure of the of the structure, a structure of GDH was proposed for and several unique segments that are in MDH of M. extorquens G.E. Anthony C. Biochem. J. PubMed Scopus Google Scholar). direct the structure has been to amino acid for GDH we mutagenesis that may be for protein GDH is a to elucidate the of catalytic reaction or intramolecular electron transfer of the primary dehydrogenase in the respiratory is the on GDH and for that showed a with on the activity K. H. Lett. 1994; 16: Scopus Google Scholar). obtained several E. coli and characterized in to their glucose ubiquinone and glucose oxidase with a pronounced on the enzyme were and their kinetic were The of mutants were further by different amino acid substitutions. at Asp-730 and related to proton transfer and to binding to PQQ, to which no was proposed in the GDH G.E. Anthony C. Biochem. J. PubMed Scopus Google Scholar). and the were from were by on a or by were of The used in were of E. coli Their and are in of the the GDH, and the from and were by in and for Escherichia coli and Scholar). The was to on glucose in and for Escherichia coli and Scholar). mutagenesis in and for Escherichia coli and Scholar) of and Jr., M.H. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), that on the glucose was and used for on in and for Escherichia coli and Scholar) glucose and PQQ. or which the as showed on the of and or and or H. Saier K. Arents J.C. Bader R. Yamada M. Adachi O. Postma P.W. Microbiology. 1997; 143: 3149-3156Crossref PubMed Scopus (85) Google M. Asaoka S. Saier Jr., M.H. Yamada Y. J. Bacteriol. 1993; 175: 568-571Crossref PubMed Google PubMed Scopus Google J. Bacteriol. PubMed Google J. J. 1987; PubMed Scopus Google with of with of with of by on by on by H775R on by and on on from on from on from on from by on by on by D730N on by on on from on from on from on from on by site-directed on from by site-directed on by site-directed on by site-directed on by site-directed on by site-directed D730A on by site-directed D730R on by site-directed H775A on by by M. by site-directed in a To the on a M. Asaoka S. Saier Jr., M.H. Yamada Y. J. Bacteriol. 1993; 175: 568-571Crossref PubMed Google Scholar), was a low of a in which the of had been with the of The from was site of The from was site of the the was site of The on is about at the region that on used these for different mutagenesis because they have different sites at the each region of the was the of to enzyme To the conserved C from the to amino acid in GDH, mutagenesis was performed with and in in a of in which a each of and and or each of and and of each and of polymerase were in and polymerase chain each of which of at for at for and at for were by a as (10Yamada M. Sumi K. Matsushita K. Adachi O. Yamada Y. J. Biol. Chem. 1993; 268: 12812-12817Abstract Full Text PDF PubMed Google Scholar). The were by and by after from the and The were and and site of To the region of GDH, in mutagenesis with was as R. G. Yamada M. Saier Jr., M.H. J. Bacteriol. 1988; 170: PubMed Google Scholar). of in was with of in and and at The were three and by The was used as a for was and on in and for Escherichia coli and Scholar) glucose, PQQ, and were as the To mutants the were by using GDH as P. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar) and by of GDH activity. T. J. Molecular Scholar) were To the was by the chain S. S. A. PubMed Scopus Google Scholar) after or C. J. J. ( ). 1985; PubMed Scopus Google Scholar) or with the United To the region including the site of the mutants from the and the was of the the C-terminal five-sixth portion of the enzyme was by showed the using from the by indicated that of have sites the and sites and the have the and regions were to To and mutagenesis was using the for for for for for and for were by or were in and for at and with The were by through a by a low membrane were by at for The membrane was in of for enzyme and of GDH, and glucose oxidase activity were as K. Shinagawa E. Adachi O. Ameyama M. J. Biochem. ( Tokyo ). 1989; 105: 633-637Crossref PubMed Scopus (47) Google Scholar, M. Sumi K. Matsushita K. Adachi O. Yamada Y. J. Biol. Chem. 1993; 268: 12812-12817Abstract Full Text PDF PubMed Google Scholar). activity was also with or of glucose, as a substrate as M. Nonobe M. Shinagawa E. Matsushita K. K. Adachi O. Biol. Chem. Scopus Google Scholar). dehydrogenase activity was as a by the used for GDH that PQQ was were from the by using the was to the and Biochem. PubMed Scopus Google Scholar) using as a were by using a of in in and in the or of was from to at with at with significant on the enzyme activity were to the as (10Yamada M. Sumi K. Matsushita K. Adachi O. Yamada Y. J. Biol. Chem. 1993; 268: 12812-12817Abstract Full Text PDF PubMed Google Scholar). and purification were performed in the of to enzyme activity. proteins as well as were by revealing that their was To fluorescence the protein was to with and after with the in the of the enzyme was with the of from the conserved C of GDH, were to activity of the the from the the to by the mutagenesis were of were to have a in the region and the remaining has and for of the mutants at the conserved amino acid among of E. A. calcoaceticus, and G. oxydans Of these three from and were by analysis using E. coli GDH but not from not has a at the in the in GDH to The protein from is to have amino acid because of of the which was indicated as a in of mutants from had activity of the mutants were also by to GDH To the region including the and the was performed as and the regions were of the or are to or C to as in suggesting that they are from the and at conserved amino acid among of the three To the with the of and glucose oxidase were with the membrane GDH protein in the were by analysis and activity was on the GDH The revealed that have activity of the with and glucose oxidase for and a The to of and glucose oxidase to of the their reduced may be to of GDH in the membrane that of the The reduced activity without the significant for PQQ and Mg2+ or on glucose oxidase which the electron transfer from GDH via to cytochrome that the C-terminal amino acid of the GDH may of the or the of the site but not of the and glucose oxidase of in the membrane and their kinetic oxidase GDH in of GDH protein in the membrane was from the of in and as a of that from the of activity were by the of GDH protein in the membrane in are of activity or activity of the in are of activity or activity of the activity was in the of glucose, PQQ, and and H775R PQQ was at a of that used for The for was performed at for were by activity. activity was at by the of glucose and after in the of PQQ and oxidase activity was at by the of glucose after in the of PQQ and and PQQ was at a of and are the of to three performed in The of GDH protein in the membrane was from the of in and as a of that from the of activity were by the of GDH protein in the membrane in are of activity or activity of the in a activity was in the of glucose, PQQ, and and H775R PQQ was at a of that used for The for was performed at for were by activity. activity was at by the of glucose and after in the of PQQ and oxidase activity was at by the of glucose after in the of PQQ and and PQQ was at a of and are the of to three performed in To of the their kinetic were with of the in the membrane and H775R showed for PQQ, and H775R had for PQQ and the showed for showed the for glucose as that of that no to be at the site. GDH has for and M. Nonobe M. Shinagawa E. Matsushita K. K. Adachi O. Biol. Chem. Scopus Google Scholar), substrate of the was but no showed different substrate from the not Therefore, is that no occurs in amino acid related to the substrate and that the in these may not of the Of significant and were to purification for further in which and H775R showed affinity for PQQ and D730N had reduced of and glucose oxidase but for PQQ, Mg2+, and glucose. and were PQQ was to used in purification suggesting that and are located the PQQ-binding site and to a of PQQ from the enzyme which may of GDH Whereas, the be in the without PQQ the The kinetic of the were to be the as obtained with the membrane showed of the and for glucose and Mg2+ to of the and for D730N were to have reduced affinity for PQQ. to these with the amino acid surrounding PQQ or Ca2+ in the structure of a GDH, which was proposed by and Anthony G.E. Anthony C. Biochem. J. PubMed Scopus Google Scholar) on the structure of the M. extorquens as The structure is with consisting of and has a to of which are specific for GDH or from of were in the of PQQ, and for was mutants and PQQ was at a of and The for was performed at for in a were in the of PQQ, and for was mutants and PQQ was at a of and The for was performed at for the the amino acid of and H775R are located close to the site in the The the affinity for PQQ but not for Mg2+, to PQQ, and and to Ca2+ in the these and the that purification the of PQQ, is that the to in may a local and also influence the of to affinity for PQQ. H775R showed reduced affinity for PQQ and To is a side by the from to or we a H775A with of a The also showed a low affinity for PQQ, but had activity to that of the that His-775 may directly interact with PQQ and that the but not the may the of the site to the assume that His-775 to the of PQQ of proposed in the and the His-775 with or to reduction in the affinity for PQQ. the may the of to to of the by site-directed in membrane GDH in the membrane was after analysis using GDH and and not not for PQQ of the were in the of glucose, The for was performed at for are the of performed in The GDH in the membrane was after analysis using GDH and and not not in a for PQQ of the were in the of glucose, The for was performed at for are the of performed in A.-M. Dekker S. van de Putte P. Goosen N. Mol. Gen. Genet. 1991; 229: 206-212Crossref PubMed Scopus (43) Google Scholar) have that in oxydans GDH, the from the conserved to His-775 in E. coli GDH, changed the substrate from glucose to the His-775 PQQ in the and the of His-775 by was proposed to a site through the G.E. Anthony C. Biochem. J. PubMed Scopus Google Scholar). revealed that the for of H775R and H775A were and which were that of the GDH, their for glucose were the as that of the Therefore, His-775 as proposed may be to the and the the mutants of G. oxydans and E. be to of the or of the in each To the of the on affinity for PQQ, a was and characterized The affinity of was similar to that of activity was that of that the with amino acid may the local structure and that may have a functional role be that a close to may be in binding to PQQ, we conserved amino acid and to three and The proteins were to be low in membrane and to be The of proteins were that of the the membrane were in the of PQQ and to for not be after and not occurs on the of G.E. Anthony C. Biochem. J. PubMed Scopus Google Scholar) and to of GDH, the is assumed to the structure via to a on appear to be different from that of or because in the of PQQ and in the were showed no on affinity for PQQ and on activity. Therefore, we the that are specific with PQQ or to catalytic these with of and that the of reduction of the affinity for PQQ by the the PQQ-binding and that the in the a local structure the in the is located the of and at the end of of which is conserved in but in MDHs or ADH G.E. Anthony C. Biochem. J. PubMed Scopus Google and that the might be for PQQ binding in for the sites of the three and are no amino acid with PQQ or Mg2+ in the occurs in the of the which is from MDH and on affinity for Mg2+ mutants Therefore, is that the site. The sites of and are located at the C-terminal region highly conserved in the quinoprotein dehydrogenase and and occur in the of the C-terminal region of the are with a tryptophan but with to such a no specific function has been in the of MDH G.E. Anthony C. Biochem. J. PubMed Scopus Google Scholar). as well as which has the C that may the structure, reduced the of the proteins in the membrane and suggesting that the conserved region is for of the enzyme region might also the site for the N-terminal ubiquinone-binding domain in GDH or GDH D730N showed of and glucose but no on affinity for PQQ or Mg2+ and The from to not to a large To further the function of D730A and were and characterized with membrane mutants also showed low to to D730N and for PQQ was in to or not the D730N the local structure the PQQ-binding we the local by of tryptophan fluorescence by of PQQ not of D730N and the the fluorescence spectrum with at at fluorescence were by the of PQQ, and the with PQQ was that the to at may not the local surrounding PQQ but the may influence the structure the PQQ-binding site to the affinity for PQQ. Asp-730 may be located close to the PQQ-binding occurs structures in of the the large in was among Asp-730 of suggesting that Asp-730 may have a catalytic these we assume that Asp-730 may close to the catalytic site and is in proton to PQQ or proton from PQQH2. GDH electrons directly to MDH to (20Anthony C. Int. J. Biochem. 1992; 24: 29-39Crossref PubMed Scopus (50) Google Scholar), and ADH to the intramolecular heme c (23Matsushita K. Yakushi T. Toyama H. Shinagawa E. Adachi O. J. Biol. Chem. 1996; 271: 4850-4857Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), they are proposed to a structure C.C.F. Ghosh M. Harlos K. Avezoux A. Anthony C. Nat. Struct. Biol. 1994; 1: 102-105Crossref PubMed Scopus (89) Google Scholar, G.E. Anthony C. Biochem. J. PubMed Scopus Google Scholar). appear to have a similar surrounding PQQ and the to each other, but to different segments or specific for the intramolecular electron transfer to the specific electron Therefore, GDH has unique segments the structure and a segment in the conserved among occurs the conserved tryptophan and where is in the structure is a and conserved the and might play a specific role for GDH from catalytic reaction to electron transfer to ubiquinone. M. and M. H. for also M. for