Structural Analysis of the fds Operon Encoding the NAD+-linked Formate Dehydrogenase of Ralstonia eutropha
1998; Elsevier BV; Volume: 273; Issue: 41 Linguagem: Inglês
10.1074/jbc.273.41.26349
ISSN1083-351X
Autores Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoThe fdsGBACD operon encoding the four subunits of the NAD+-reducing formate dehydrogenase of Ralstonia eutropha H16 was cloned and sequenced. Sequence comparisons indicated a high resemblance of FdsA (α-subunit) to the catalytic subunits of formate dehydrogenases containing a molybdenum (or tungsten) cofactor. The NH2-terminal region (residues 1–240) of FdsA, lacking in formate dehydrogenases not linked to NAD(P)+, exhibited considerable similarity to that of NuoG of the NADH:ubiquinone oxidoreductase from Escherichia colias well as to HoxU and the NH2-terminal segment of HndD of NAD(P)+-reducing hydrogenases. FdsB (β-subunit) and FdsG (γ-subunit) are closely related to NuoF and NuoE, respectively, as well as to HoxF and HndA. It is proposed that the NH2-terminal domain of FdsA together with FdsB and FdsG constitute a functional entity corresponding to the NADH dehydrogenase (diaphorase) part of NADH:ubiquinone oxidoreductase and the hydrogenases. No significant similarity to any known protein was observed for FdsD (δ-subunit). The predicted product of fdsC showed the highest resemblance to FdhD from E. coli, a protein required for the formation of active formate dehydrogenases in this organism. Transcription of the fdsoperon is subject to formate induction. A promoter structure resembling the consensus sequence of ς70-dependent promoters from E. coli was identified upstream of the transcriptional start site determined by primer extension analysis. The fdsGBACD operon encoding the four subunits of the NAD+-reducing formate dehydrogenase of Ralstonia eutropha H16 was cloned and sequenced. Sequence comparisons indicated a high resemblance of FdsA (α-subunit) to the catalytic subunits of formate dehydrogenases containing a molybdenum (or tungsten) cofactor. The NH2-terminal region (residues 1–240) of FdsA, lacking in formate dehydrogenases not linked to NAD(P)+, exhibited considerable similarity to that of NuoG of the NADH:ubiquinone oxidoreductase from Escherichia colias well as to HoxU and the NH2-terminal segment of HndD of NAD(P)+-reducing hydrogenases. FdsB (β-subunit) and FdsG (γ-subunit) are closely related to NuoF and NuoE, respectively, as well as to HoxF and HndA. It is proposed that the NH2-terminal domain of FdsA together with FdsB and FdsG constitute a functional entity corresponding to the NADH dehydrogenase (diaphorase) part of NADH:ubiquinone oxidoreductase and the hydrogenases. No significant similarity to any known protein was observed for FdsD (δ-subunit). The predicted product of fdsC showed the highest resemblance to FdhD from E. coli, a protein required for the formation of active formate dehydrogenases in this organism. Transcription of the fdsoperon is subject to formate induction. A promoter structure resembling the consensus sequence of ς70-dependent promoters from E. coli was identified upstream of the transcriptional start site determined by primer extension analysis. formate dehydrogenase soluble NAD+-linked formate dehydrogenase molybdopterin guanine dinucleotide kilobase pair(s) base pair(s) NADH:ubiquinone oxidoreductase. Apart from molecular hydrogen, formate can serve as an alternative energy source for autotrophic growth of the aerobic, facultatively chemoautotrophic bacterium Ralstonia eutropha (formerlyAlcaligenes eutrophus) (1Bowien B. Schlegel H.G. Annu. Rev. Microbiol. 1981; 35: 405-452Crossref PubMed Scopus (191) Google Scholar). The oxidation of formate in this organism is catalyzed by two distinct types of FDH1: a soluble, NAD+-linked enzyme (S-FDH; EC 1.2.1.2) and a membrane-bound enzyme coupled directly to the respiratory chain via an unknown electron acceptor (2Friedrich C.G. Bowien B. Friedrich B. J. Gen. Microbiol. 1979; 115: 185-192Crossref Scopus (83) Google Scholar). S-FDH catalyzes the irreversible oxidation of formate to CO2 with concomitant reduction of NAD+ to NADH. Assimilation of CO2 proceeds via the reactions of the reductive pentose phosphate cycle (3Bowien B. Windhövel U. Yoo J.-G. Bednarski R. Kusian B. FEMS Microbiol. Rev. 1990; 87: 445-450Crossref Scopus (13) Google Scholar). S-FDH exhibits diaphorase activity by reducing electron acceptors such as methyl viologen, benzyl viologen, or ferricyanide with NADH as electron donor (4Friedebold J. Bowien B. J. Bacteriol. 1993; 175: 4719-4728Crossref PubMed Google Scholar). Therefore, the enzyme has two distinct activities, a FDH and a NADH dehydrogenase activity, which combine to perform the complete catalytic reaction. The enzyme is composed of four nonidentical subunits (αβγδ) and contains one molecule of each MGD and FMN in addition to a number of redox-active [Fe-S] centers as cofactors (4Friedebold J. Bowien B. J. Bacteriol. 1993; 175: 4719-4728Crossref PubMed Google Scholar, 5Friedebold J. Mayer F. Bill E. Trautwein A.X. Bowien B. Biol. Chem. Hoppe-Seyler. 1995; 376: 561-568Crossref PubMed Scopus (19) Google Scholar). Based on their general structure, FDHs can be divided into two groups. The first group of enzymes comprising heteromeric FDHs with various physiological functions is characterized by the possession of molybdenum or tungsten cofactors and [Fe-S] centers. Their catalytic subunits show significant sequence similarity. Depending on the physiological function of the individual enzymes, structures and cofactor contents of the remaining subunits are more diverse (6Ferry J.G. FEMS Microbiol. Rev. 1990; 87: 377-382Crossref Scopus (101) Google Scholar). The S-FDH from R. eutropha belongs to this group together with FDHs from various bacterial and archaeal organisms such asEscherichia coli, Wolinella succinogenes, Moorella thermoacetica (formerly Clostridium thermoaceticum) and Methanobacterium formicicum. The second group represents the homodimeric, NAD+-reducing FDH from methylotrophic bacteria and yeasts and from plants. These enzymes contain neither cofactors nor metals. Their amino acid sequences resemble considerably and are also similar to those of NAD+-dependent, d-specific 2-hydroxyacid dehydrogenases like lactate dehydrogenase (7Popov V.O. Lamzin V.S. Biochem. J. 1994; 301: 625-643Crossref PubMed Scopus (231) Google Scholar). The enzymes of the two groups share very little similarity except for a short sequence region. The present work reports on the first cloning and sequencing of genes encoding a heteromeric FDH from an aerobic, autotrophic organism. Thefds genes from R. eutropha H16 apparently form an operon consisting of the four structural genes of S-FDH (fdsA, fdsB, fdsG, and fdsD) and an additional gene (fdsC) of unknown function not related to the enzyme. Analysis of the deduced amino acid sequences enabled a prediction of the cofactor sites within the S-FDH subunits and allowed us to hypothesize on a path of intramolecular electron transfer. Furthermore, structural relationships between the subunits of S-FDH and FDHs from other organisms are discussed, and similarities to subunits of NAD+/NADP+-reducing hydrogenases and NADH:ubiquinone oxidoreductases (complex I) are unveiled. Transcriptional studies are also presented, aimed at the regulation and promoter identification of the operon. The bacterial strains, phages, and plasmids used in this study are listed in Table I. R. eutropha was grown under air at 30 °C in a mineral salts medium supplemented with either 0.2% (w/v) formate (autotrophic growth), 0.2% (w/v) fructose (heterotrophic growth), or 0.2% (w/v) formate plus 0.1% (w/v) fructose (mixotrophic growth) as described previously (4Friedebold J. Bowien B. J. Bacteriol. 1993; 175: 4719-4728Crossref PubMed Google Scholar). Alternatively, hydrogen and CO2 (8:1, v/v) were used as energy and carbon sources, respectively, for aerobic lithoautotrophic growth of the organism. Cultures to be employed in RNA isolation were grown in low phosphate mineral medium (11Kusian B. Bednarski R. Husemann M. Bowien B. J. Bacteriol. 1995; 177: 4442-4450Crossref PubMed Google Scholar) to an optical density of 2–3 (mid-exponential phase) measured at 436 nm. Strains of E. coli were cultivated aerobically at 37 °C in Luria-Bertani medium. If required, ampicillin was added to the medium at a concentration of 50 μg/ml. Phages were propagated in E. coli WL87 or WL95 as host strains according to Sambrook et al. (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).Table IBacterial and phage strains and plasmids used in this workStrain or plasmidRelevant phenotype or genotypeSource or Ref.StrainsR. eutropha H16Wild type; Cfx, Hox, FoxATCC 17699E. coliXL1-BluerecA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, F′[proAB, lacIZΔM15, Tn10(Tcr)]8Bullock W.O. Fernandez J.M. Short J.M. BioTechniques. 1987; 5: 376-378Google ScholarE. coliWL87recBCAmershamE. coliWL95recBC, P2 lysogenAmershamPhages and plasmidsλL47srIλ1–2, imm434 cI, nin5, chiA1319Loenen W.A.M. Brammar W.J. Gene (Amst.). 1980; 10: 249-259Crossref PubMed Scopus (227) Google ScholarλAEC6λL47::12-kbBamHI fragment; phage clone from a partial genomic library of strain H16 with inserted fragment carrying the fdsoperonThis studypUC18Apr; lacPOZ′10Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (12033) Google ScholarpBluescript SK+Apr; lacPOZ′StratagenepOH1–1pUC18::12-kb BamHI fragment from λAEC6This studypOH1–2pUC18::12-kb BamHI fragment from λAEC6This studypSOH1–1pBluescript SK+::3.1-kbEcoRI fragment from pOH1–1This studypSOH1–2pBluescript SK+::3.1-kbEcoRI fragment from pOH1–1This studypSOH2pBluescript SK+::2.9-kbEcoRI-XhoI fragment from pOH1–1This studypSOH5pBluescript SK+::3.2-kbSmaI-XhoI fragment from pOH1–1This studyCfx, ability for autotrophic CO2 fixation; Hox, ability for H2 oxidation; Fox, ability for formate oxidation; Tcr, tetracycline resistance; Apr, ampicillin resistance.The last digit of the plasmid designations refers to the transcriptional orientation of the cloned fds operon relative to lacZ′ of the vectors (1, colinear; 2, divergent). Open table in a new tab Cfx, ability for autotrophic CO2 fixation; Hox, ability for H2 oxidation; Fox, ability for formate oxidation; Tcr, tetracycline resistance; Apr, ampicillin resistance. The last digit of the plasmid designations refers to the transcriptional orientation of the cloned fds operon relative to lacZ′ of the vectors (1, colinear; 2, divergent). Genomic DNA fromR. eutropha H16 was isolated according to Ausubel et al. (13Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, New York1988: 2.4.1-2.4.5Google Scholar). Plasmid DNA from E. coli XL-1 Blue was prepared by alkaline lysis of cells as detailed by Sambrook et al. (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and, if necessary, purified further using special chromatographic columns (Qiagen, Hilden, Germany). Recombinant DNA manipulations were performed by standard procedures (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For double-stranded sequence analysis, nested deletions in pSOH1-1, pSOH1-2, pSOH2, and pSOH5 were generated by treatment with exonuclease III and mung bean nuclease according to the instructions of the manufacturer (Stratagene, Heidelberg, Germany). Nucleotide sequences were determined by the cycle sequencing method employing a reagent kit (SequiTherm Cycle Sequencing Kit; Biozym, Hessisch Oldendorf, Germany) together with either 35S- or fluorescence-labeled oligonucleotide primers. The oligonucleotides were purchased from or synthesized by Pharmacia (Freiburg, Germany) or MWG-Biotech (Ebersberg, Germany), respectively. Nucleotide and deduced amino acid sequences were analyzed by the latest version of the GCG program package (14Devereux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (12575) Google Scholar). Multiple alignments of sequences were constructed by means of the programs ClustalW (15Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56668) Google Scholar) or MACAW, version 2.0.0 (16Lawrence C.E. Altschul S.F. Boguski M.S. Liu J.S. Neuwald A.F. Wotton J.C. Science. 1993; 262: 208-213Crossref PubMed Scopus (1461) Google Scholar). For Southern hybridizations, restriction fragments of DNA were separated by agarose gel electrophoresis and transferred onto a nylon membrane (Biodyne B; Pall, Dreieich, Germany) using a vacuum blotting device (Vacu-Gene XL; Pharmacia). Labeling of DNA probes, hybridization, and signal detection were carried out using the ECL 3′-Oligolabeling and Detection System as instructed by the manufacturer (Amersham Buchler, Brunswick, Germany). Genomic DNA (300 μg) from R. eutropha H16 was digested to completion with 700 units of restriction endonuclease BamHI. To isolate fragments of the 8–20-kb size range, the digested DNA was electroeluted from the corresponding gel area after agarose (0.8%, w/v) gel electrophoresis in Tris acetate buffer, pH 8.1. The fractionated DNA was then ligated into the BamHI site of vector phage λL47 and subjected to in vitro packaging using the Gigapack II Gold Packaging Extract (Stratagene). The resulting phage particles representing a partial genomic library of strain H16 were initially propagated in E. coli WL95 and subsequently screened by plaque hybridization after infection of E. coli WL87. Labeling of the oligonucleotide probes specific for fds genes and signal detections were performed employing the ECL 3′-Oligolabeling and Detection System. Total RNA was isolated fromR. eutropha H16 as described by Oelmüller et al. (17Oelmüller U. Krüger N. Steinbüchel A. Friedrich C.G. J. Microbiol. Methods. 1990; 11: 73-84Crossref Scopus (121) Google Scholar). For Northern hybridization experiments, denatured RNA (20 μg/lane) was applied to a formaldehyde agarose gel, separated by electrophoresis, and transferred onto a nylon membrane (Biodyne B) by vacuum blotting. DNA probes used in RNA hybridizations were labeled radioactively with [α-32P]dCTP by means of a random primer labeling system (Life Technologies, Eggenstein, Germany). A 30-mer oligonucleotide primer complementary to nucleotide positions 30–59 downstream of the translational start of the fdsG gene was radioactively labeled at its 5′end using [γ-33P]ATP (NEN, Bad Homburg, Germany) and T4 polynucleotide kinase (Life Technologies). In a volume of 10 μl of 50 mm Tris-HCl buffer, pH 8.3, containing 55 mm KCl, 3 mm MgCl2, and 12.5 units of RNase inhibitor, 20 μg of total RNA from R. eutropha H16 was denatured at 80 °C for 5 min and annealed at 37 °C for 3 h with 0.2 pmol of the labeled primer. The annealed primer was extended at 37 °C for 1 h in 50 μl of 50 mm Tris-HCl, pH 8.3, containing 55 mm KCl, 3 mm MgCl2; 0.5 μg of actinomycin D; 10 mm dithiothreitol; 0.5 mm each dATP, dCTP, dGTP and dTTP; and 12.5 units of RNase inhibitor, in the presence of 200 units of reverse transcriptase (Pharmacia). The extended products were precipitated with ethanol after the addition of 3.5 μg of salmon sperm DNA, redissolved in 5 μl of H2O, and finally analyzed by denaturing polyacrylamide gel electrophoresis (13Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, New York1988: 2.4.1-2.4.5Google Scholar). To determine the sizes of these products, the same oligonucleotide was used as primer in a sequencing reaction with pSOH1-1. Autoradiography was done using either Hyperfilm β-max (Amersham Buchler) or Cronex 10S film (NEN). The approach to clone the S-FDH genes of R. eutrophawas based on the known NH2-terminal amino acid sequences of the four subunits of the enzyme (5Friedebold J. Mayer F. Bill E. Trautwein A.X. Bowien B. Biol. Chem. Hoppe-Seyler. 1995; 376: 561-568Crossref PubMed Scopus (19) Google Scholar). Southern blotting revealed that a 12-kb BamHI fragment of the chromosomal DNA from R. eutropha H16 hybridized with degenerate oligonucleotide probes deduced from these sequences (data not shown). Phage clone λAEC6 containing the fragment was isolated by screening a partial genomic library of strain H16 using the α- and δ-probes. After subcloning of the fragment into pUC18 in both orientations relative tolacZ′ (pOH1-1 and pOH1-2), the relative positions of the S-FDH structural genes (fds genes) within the fragment (Fig. 1) were determined by restriction analysis and Southern hybridization employing all four oligonucleotides as probes. A genomic segment of 6,589 bp was sequenced and found to contain a cluster of five colinearly oriented open reading frames that were designated as genes fdsG (531 bp), fdsB (1,563 bp), fdsA (2,880 bp), fdsC (861 bp), and fdsD (384 bp) (Fig. 1). Start (ATG) and stop codons (TGA) of the genes either overlap (fdsGB, fdsCD) or are separated by 15- (fdsAC) or 35-bp (fdsBA) intergenic regions. The codon usage agrees well with that of knownR. eutropha genes. The fds genes are thus likely to form a pentacistronic operon. A comparison of the deduced amino acid sequences with the previously determined NH2-terminal amino acid sequences of the S-FDH subunits (5Friedebold J. Mayer F. Bill E. Trautwein A.X. Bowien B. Biol. Chem. Hoppe-Seyler. 1995; 376: 561-568Crossref PubMed Scopus (19) Google Scholar) revealed that fdsG, fdsB, fdsA, and fdsD represent the structural genes of the four subunits γ, β, α, and δ, respectively. The calculated isoelectric points of the putative gene products were in the weakly acidic range for FdsA (pI = 6.77), FdsB (5.44), FdsC (6.99), and FdsG (6.32), whereas it was remarkably basic (pI = 9.86) for FdsD. A hydropathy analysis (18Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (18256) Google Scholar) of the five proteins suggested that none of them contains potential membrane-spanning helices and hydrophilic and hydrophobic residues to be distributed evenly within the polypeptide chains (data not shown), confirming the cytoplasmic location of S-FDH. FdsA is the largest subunit of S-FDH from R. eutropha and consists of 959 residues with a calculated molecular mass of 105 kDa, which agrees well with that determined previously by SDS-polyacrylamide gel electrophoresis (4Friedebold J. Bowien B. J. Bacteriol. 1993; 175: 4719-4728Crossref PubMed Google Scholar). It possesses high sequence similarity (51–62%) to the catalytic subunits (α-subunits) of FDHs from various prokaryotes like M. thermoacetica (19Li, X.-L., Ljungdahl, L. G., and Gollin, D. J. (1996) GenBank/EMBL/DDBJ accession number U73807Google Scholar),M. formicicum (20Shuber A.P. Orr E.C. Recny M.A. Schendel P.F. May H.D. Schauer N.L. Ferry J.G. J. Biol. Chem. 1986; 261: 12942-12947Abstract Full Text PDF PubMed Google Scholar), E. coli (21Zinoni F. Birkmann A. Stadtman T.C. Böck A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4650-4654Crossref PubMed Scopus (372) Google Scholar, 22Berg B.L. Li J. Heider J. Stewart V. J. Biol. Chem. 1991; 266: 22380-22385Abstract Full Text PDF PubMed Google Scholar, 23Abaibou H. Pommier J. Benoit S. Giordano G. Mandrand-Berthelot M.-A. J. Bacteriol. 1995; 177: 7141-7149Crossref PubMed Scopus (83) Google Scholar), and W. succinogenes (24Bokranz M. Gutmann M. Körtner C. Kojro E. Fahrenholz F. Lauterbach F. Kröger A. Arch. Microbiol. 1991; 156: 119-128Crossref PubMed Scopus (71) Google Scholar, 25Lenger R. Herrmann U. Gross R. Simon J. Kröger A. Eur. J. Biochem. 1997; 246: 646-651Crossref PubMed Scopus (26) Google Scholar) which contain MGD as molybdenum cofactor or a tungsten cofactor in the case of FDH from M. thermoacetica (26Yamamoto I. Saiki T. Liu S.-M. Ljungdahl L.G. J. Biol. Chem. 1983; 258: 1826-1832Abstract Full Text PDF PubMed Google Scholar, 27Kletzin A. Adams M.W.W. FEMS Microbiol. Rev. 1996; 18: 5-63Crossref PubMed Google Scholar), indicating that the α-subunit of R. eutropha S-FDH catalyzes the oxidation of formate. The hypothetical flpF gene product of Methanobacterium thermoautotrophicum (28Hochheimer, A., Schmitz, R. A., Thauer, R. K., and Hedderich, R. (1995) GenBank/EMBL/DDBJ accession number X87969Google Scholar) also exhibited such a high degree of resemblance (61% similarity). In contrast, except for a short sequence region (see below), no similarity between FdsA and homodimeric, NAD+-reducing FDH from either methylotrophic bacteria (29Popov V.O. Shumilin I.A. Ustinnikova T.B. Lamzin V.S. Egorov T.A. Bioorg. Khimia (U. S. S. R.). 1990; 16: 324-335PubMed Google Scholar,30Galkin A. Kulakova L. Tishkov V. Esaki N. Soda K. Appl. Microbiol. Biotechnol. 1995; 44: 479-483Crossref PubMed Scopus (64) Google Scholar), yeasts (31Hollenberg, C. P., and Janowicz, Z. (1989) GenBank/EMBL/DDBJ accession number A06214Google Scholar, 32Allen S.J. Holbrook J.J. Gene (Amst.). 1995; 162: 99-104Crossref PubMed Scopus (37) Google Scholar), or a plant (33Colas des Francs-Small C. Ambard-Bretteville F. Small I.D. Rémy R. Plant Physiol. 1993; 102: 1171-1177Crossref PubMed Scopus (103) Google Scholar) was found. A multiple sequence alignment of FdsA and related proteins (Fig. 2) revealed that eight regions (C5, O1, F1, O2, M1, F2, O3, and M2) are conserved in the α-subunits of all MGD-containing FDHs a well as in the periplasmic nitrate reductase of R. eutropha (34Siddiqui R.A. Warnecke-Eberz U. Hengsberger A. Schneider B. Kostka S. Friedrich B. J. Bacteriol. 1993; 175: 5867-5876Crossref PubMed Google Scholar). The F1 and F2 regions are well conserved only in the FDHs and in the periplasmic nitrate reductase, but not in other molybdopterin cofactor-containing oxidoreductases (biotin sulfoxide reductase, dimethyl sulfoxide reductase, polysulfide reductase, trimethylamine N-oxide reductase; 35–38). The selenocysteine residue essential for catalytic activity of the FDH isoenzymes FDH-H and FDH-N from E. coli (Fig. 3 A) and of the tungsten-containing enzyme of M. thermoacetica (not shown) is located in the F1 region. In the other FDHs and in the periplasmic nitrate reductase cysteine (Cys378 in FdsA) replaces selenocysteine. Selenium and sulfur, respectively, are the proposed ligands of the molybdenum in MGD (42Gladyshev V.N. Khangulov S.V. Axley M.J. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7708-7711Crossref PubMed Scopus (85) Google Scholar). The neighboring histidine residue (His379 in FdsA) is conserved in all MGD-containing FDHs and plays a role in orienting the substrate molecule formate and in proton abstraction from formate during catalysis (43Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (508) Google Scholar). It seems conceivable that the enzymes containing cysteine instead of selenocysteine have a different catalytic mechanism, a presumption supported by the fact that FDH from M. formicicum has a sulfido group as a ligand of molybdenum (Mo=S; 44), which does not occur in FDH-H (43Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (508) Google Scholar). The sulfido group, instead of the selenol group, possibly serves as a proton acceptor during the transfer of a hydride ion from formate to the molybdenum cofactor (45Hille R. Chem. Rev. 1996; 96: 2757-2856Crossref PubMed Scopus (1496) Google Scholar). It has been shown that S-FDH of R. eutropha is inactivated irreversibly by cyanide (4Friedebold J. Bowien B. J. Bacteriol. 1993; 175: 4719-4728Crossref PubMed Google Scholar) much in the same manner as FDH from M. formicicum and the members of the xanthine oxidase family containing the Mo=S group. Cyanide inactivates molybdoenzymes by replacing Mo=S with Mo=O to yield the inactive desulfo form of the enzymes (45 and references therein). Furthermore, mutated FDH-H fromE. coli, in which cysteine replaces selenocysteine, showed a 300 times lower activity than the wild-type enzyme (46Axley M.J. Böck A. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8450-8454Crossref PubMed Scopus (186) Google Scholar). The recent determination of the crystal structures of dimethyl sulfoxide reductase from Rhodobacter sphaeroides and FDH-H from E. coli revealed in both enzymes two MGD molecules to be involved in the coordination of one molybdenum atom by means of four dithiolene ligands (43Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (508) Google Scholar, 47Schindelin H. Kisker C. Hilton J. Rajagopalan K.V. Rees D.C. Science. 1996; 272: 1615-1621Crossref PubMed Scopus (445) Google Scholar). In discord with these findings, quantitation of MGD yielded 0.71 mol/mol of R. eutropha S-FDH, indicating that this enzyme contains only one molecule of molybdenum cofactor (4Friedebold J. Bowien B. J. Bacteriol. 1993; 175: 4719-4728Crossref PubMed Google Scholar), although the catalytic subunits of all MGD-containing FDHs share high sequence similarity. Thus, the catalytic core and mechanism of S-FDH appear to be distinct from those of FDH-H. It is proposed that S-FDH, despite the high degree of sequence similarity to FDH-H, contains only one MGD and at least one sulfido group in the coordination sphere of molybdenum.Figure 3Alignment of FdsA with various FDH subunits:panel A, amino acid residues 238–927 of FdsA; panel B, F2 region. Conserved regions (C5, O1–O3, F1 and F2, M1 and M2) are underlined, and identical or conservatively substituted residues are highlighted by a blackor gray background. Region C5 is predicted to coordinate a [2Fe-2S] center in FdsA and does so in NuoG, but it coordinates a [4Fe-4S] center in FdhF (see "Results and Discussion"). The selenocysteine residue (U) within the F1 region of FdhF and FdhG as well as the arginine in position 284 of the FDH fromPseudomonas sp. 101 (Fdh P. sp.) (7Popov V.O. Lamzin V.S. Biochem. J. 1994; 301: 625-643Crossref PubMed Scopus (231) Google Scholar) are marked by arrows. Gaps introduced to optimize the alignment are indicated by dashes. The numbers on theright give the positions of the respective residues in the proteins. The sequence similarities (in percent) between FdsA and the other proteins are indicated on the right of the last position numbers. For abbreviations of the proteins, see Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Alignment of FdsA with various FDH subunits:panel A, amino acid residues 238–927 of FdsA; panel B, F2 region. Conserved regions (C5, O1–O3, F1 and F2, M1 and M2) are underlined, and identical or conservatively substituted residues are highlighted by a blackor gray background. Region C5 is predicted to coordinate a [2Fe-2S] center in FdsA and does so in NuoG, but it coordinates a [4Fe-4S] center in FdhF (see "Results and Discussion"). The selenocysteine residue (U) within the F1 region of FdhF and FdhG as well as the arginine in position 284 of the FDH fromPseudomonas sp. 101 (Fdh P. sp.) (7Popov V.O. Lamzin V.S. Biochem. J. 1994; 301: 625-643Crossref PubMed Scopus (231) Google Scholar) are marked by arrows. Gaps introduced to optimize the alignment are indicated by dashes. The numbers on theright give the positions of the respective residues in the proteins. The sequence similarities (in percent) between FdsA and the other proteins are indicated on the right of the last position numbers. For abbreviations of the proteins, see Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The COOH-terminal half of the F2 region in these FDHs exhibited significant local similarity to a corresponding stretch in the NAD+-reducing, homodimeric FDH from Pseudomonassp. 101, where a catalytically essential arginine (Arg284in FDH of Pseudomonas sp. 101, Arg579 in FdsA) is located (Fig. 3 B). It was demonstrated by x-ray crystallography with FDH-H (FdhF) of E. coli (43Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (508) Google Scholar) and FDH of Pseudomonas sp. 101 (7Popov V.O. Lamzin V.S. Biochem. J. 1994; 301: 625-643Crossref PubMed Scopus (231) Google Scholar) that this residue forms a hydrogen bond with formate in the active site, in FDH-H together with the above mentioned histidine. Both F1 and F2 thus seem to be parts of the active center of FDHs. The fact that the two regions are also present in periplasmic nitrate reductase suggests a structurally similar active center to occur in this protein. Nitrate, the substrate of periplasmic nitrate reductase, is regarded as a structural analog of formate in the transition state during catalysis by FDH (7Popov V.O. Lamzin V.S. Biochem. J. 1994; 301: 625-643Crossref PubMed Scopus (231) Google Scholar). The C5 region includes the sequence motif Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Xaa26–34-Cys (see Figs. 2and 3 A, Fig. 4). It occurs in all FDHs and in some molybdopterin cofactor-containing oxidoreductases (periplasmic nitrate reductase, dimethyl sulfoxide reductase, polysulfide reductase; Refs. 34Siddiqui R.A. Warnecke-Eberz U. Hengsberger A. Schneider B. Kostka S. Friedrich B. J. Bacteriol. 1993; 175: 5867-5876Crossref PubMed Google Scholar, 36Bilous P.T. Cole S.T. Anderson W.F. Weiner J.H. Mol. Microbiol. 1988; 2: 785-795Crossref PubMed Scopus (131) Google Schol
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