Molybdopterin adenine dinucleotide and molybdopterin hypoxanthine dinucleotide in formylmethanofuran dehydrogenase from <i>Methanobacterium thermoautotrophicum</i> (Marburg)

Formylmethanofuran dehydrogenases, which are found in methanogenic Archaea, are molybdenum or tungsten iron-sulfur proteins containing a pterin cofactor. We report here on differences in substrate specificity, EPR properties and susceptibility towards cyanide inactivation of the enzymes from Methanosarcina barkeri, Methanobacterium thermoautotrophicum and Methanobacterium woljei.The molybdenum enzyme from M. barkeri (relative activity with N-formylmethanofuran = 100%) was found to catalyze, albeit at considerably reduced apparent V,,,,,, the dehydrogenation of N-furfurylformamide (11 %), N-methylformamide (0.2%), formamide (0.1 %) and formate (1 %). The molybdenum enzyme from M. wolfei could only use N-furfurylformamide (1 %) and formate (3 %) as pseudosubstrates. The molybdenum enzyme from M. thermoautotrophicum and the tungsten enzymes from M. thermoautotrophicum and M. wolfei were specific for N-formylmethanofuran.The molybdenum formylmethanofuran dehydrogenases exhibited at 77 K two rhombic EPR signals, designated FMDred and FMD,,, both derived from Mo as shown by isotopic substitution with 9 7 M~. The FMD,, signal was only displayed by the active enzyme in the reduced form and was lost upon enzyme oxidation; the FMD,, signal was displayed by an inactive form and was not quenched by 0,. The tungsten isoenzymes were EPR silent.The molybdenum formylmethanofuran dehydrogenases were found to be inactivated by cyanide whereas the tungsten isoenzymes, under the same conditions, were not inactivated. Inactivation was associated with a characteristic change in the molybdenum-derived EPR signal. Reactivation was possible in the presence of sulfide. Abbreviations. FMD,,,, EPR signal attributed to active formylmethanofuran dehydrogenase ; FMD,,, EPR signal attributed to inactive formylmethanofuran dehydrogenase ; R, Land6 splittina + co2 t P IE" = -497 mV (Thauer, 1990) factor; gx, gy-and g,, Land6 splitting factors in the x , y and directions for anisotropic systems; 1 U = 1 Fmol formylmethanofuran dehydrogenatedmin.The physiological electron accePtor/donor is not knownMethylviologen (E"' = -446 mV) can be used as artificial

Section: Methodsmentioning

confidence: 99%

Formylmethanofuran dehydrogenases from methanogenic Archaea Substrate specificity, EPR properties and reversible inactivation by cyanide of the molybdenum or tungsten iron‐sulfur proteins

Bertram

Karrasch

Schmitz

et al. 1994

“…Purified formylmethanofuran dehydrogenase from M . thermoautotruphicum was shown to contain 0.3 -0.6 mol molybdenum/mol enzyme, 4 mol non-heme iron/mol enzyme and 4 mol acid-labile sulfur/mol enzyme [27], and the enzyme from M. barkeri contained 0.5-0.8 mol molybdenum/mol enzyme, 28 2 mol non-heme iron/mol enzyme and 28…”

Section: Purification and Molecular Propertiesmentioning

confidence: 98%

“…Purified formylmethanofuran dehydrogenase from M . thermoautotrophicum is composed of two different subunits (60 kDa and 45 kDa) [27] and the enzyme from M . barkeri is composed of six different subunits (65,50,37,34,29 and 17 kDa) [26].…”

Section: Purification and Molecular Propertiesmentioning

confidence: 99%

See 1 more Smart Citation

A tungsten‐containing active formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei

Schmitz

Richter

Linder

et al. 1992

Methanobacterium wolfei is a thermophilic methanogenic archaeon which requires tungsten or molybdenum for growth. We have found that the organism contains two formylmethanofuran dehydrogenases, one of which is a tungsten enzyme. Indirect evidence indicates that the other formylmethanofuran dehydrogenase is a molybdenum enzyme. The tungsten enzyme was purified and characterized. The native enzyme had an apparent molecular mass of 130 kDa. SDSjPAGE revealed a composition of three subunits of apparent molecular mass 35, 51 and 64 kDa, the Nterminal amino acid sequences of two of which were determined. 0.3 -0.4 mol tungsten/mol enzyme was found but no molybdenum. The pterin cofactor was identified as molybdopterin guanine dinucleotide. The purified enzyme exhibited a specific activity of 8.3 pmol . min-' . mg protein-' and an apparent K, for formylmethanofuran and methylviologen of 13 pM and 0.4 mM, respectively.The optimum temperature for activity was 65°C. At 40 -6O"C, the rate increased with a Qlo of 1.9; the activation energy of the reaction was 45 kJ/mol. The enzyme was found to require potassium ions for thermostability. The oxygen-sensitive enzyme was not inactivated by cyanide.

“…6). In procaryotic cells the dinucleotides contain adenosine (Borner et al, 1991), guanosine (Karrasch et al, 1990), cytosine (Johnson et al, 1990) or hypoxanthine (Borner et al, 1991). As shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

The (2R)‐hydroxycarboxylate‐viologen‐oxidoreductase from Proteus vulgaris is a molybdenum‐containing iron‐sulphur protein

Trautwein

Krauss

Lottspeich

et al. 1994

An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2‐oxocar‐boxylates at the expense of reduced artificial redox mediators to (2R)‐hydroxycarboxylates has been purified to a specific activity of up to 1800 μmol · min−1· mg−1 for the reduction of phenylpyruvate. The membrane‐bound non‐pyridine nucleotide‐dependent enzyme appears in the form of various oligomers of the 80‐kDa monomer. The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on 99Mo‐labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis. Permanganate oxidation delivers 0.7 mol pterin‐6‐carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron‐sulphur clusters.