Salt dependence, kinetic properties and catalytic mechanism of <i>N</i>‐formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile <i>Methanopyrus kandleri</i>

Self Cite

Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H 4 MPT) with NAD 1 and NADP 1. The purification of a 32-kDa NADP-specific methylene H 4 MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H 4 MPT dehydrogenase (MtdB) from this aerobic a-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H 4 MPT to methenyl H 4 MPT with NAD 1 and NADP 1 via a ternary complex catalytic mechanism. The K m for methylene H 4 MPT was 50 mm with NAD 1 (V max 1100 U´mg 21) and 100 mm with NADP 1 (V max 950 U´mg 21 ). The K m value for NAD 1 was 200 mm and for NADP 1 20 mm. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal aminoacid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.Keywords: C 1 -metabolism; methylotrophic bacteria; methanogenic archaea; tetrahydromethanopterin; tetrahydrofolate.The methylotrophic a-proteobacterium Methylobacterium extorquens AM1 possesses tetrahydrofolate (H 4 F) and dephospho tetrahydromethanopterin (H 4 MPT), two structural analogue C 1 carriers [1,2]. In cell extracts of this aerobic a-proteobacterium H 4 MPT-and H 4 F-dependent enzymes were found. It was proposed that they participate in two separate C 1 pathways, the one with the H 4 MPT-dependent enzymes being mainly involved in formaldehyde oxidation to CO 2 [1,2].Three [2]. MchA and FfsA were found to have high sequence similarity to the respective enzymes from methanogenic and sulfate reducing archaea and their encoding genes were shown to be located within a cluster of genes whose deduced gene products show, with an exception of OrfX, high sequence similarity to proteins found until now only in methanogenic and sulfate reducing archaea [1]. The gene encoding MtdA is not located within this gene cluster and MtdA does not show significant sequence similarity at the protein level to archaea specific F 420 -dependent or H 2 -forming methylene H 4 MPT dehydrogenases [4]. MtdA shows only minor sequence identity (, 15%) to methylene H 4 F dehydrogenases from bacteria and eucarya [5±9], which is consistent with the fact that ...

“…Tetrahydromethanopterin (H 4 MPT) and methenyl H 4 MPT were purified from Methanobacterium thermoautotrophicum strain Marburg (DSM 2133) [11]. Tetrahydrofolic acid (H 4 F) was from Sigma.…”

Section: A T E R I a L S A N D M E T H O D Smentioning

confidence: 99%

Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1

Hagemeier

Chistoserdova

Lidstrom

et al. 2000

Self Cite

“…CH,O was prepared from paraformaldehyde by hydrolysis for 12 h at 70°C. Tetrahydromethanopterin (H,MPT) and CH=H,MPT' were isolated from Methanobacteriurn therrnoautotrophicum strain Marburg (Breitung et al, 1992). CH2 = H,MPT was formed from H,MPT and CH,O by spontaneous reaction (Escalante-Semerena et al, 1984).…”

Section: Methodsmentioning

confidence: 99%

Hydrogen Isotope Effects in the Reactions Catalyzed by H₂‐forming N⁵,N¹⁰‐Methylenetetrahydromethanopterin Dehydrogenase from Methanogenic Archaea

Klein

Hartmann

Thauer

1995

Self Cite

H,-forming N',N"'-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron-sulfur clusters, catalyzes the reversible reduction of N5,N"'-methenyltetrahydomethanopterin (CH=H,MPT.') with H, to N',N"'-methylenetetrahydromethanopterin (CH,=H,MPT) and a proton (AGO' = -5.5 kJ/mol). The enzyme also catalyzes a CH-H,MPT+-dependent H,/H' exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH=H,MPT' reduction was performed with DZ instead of H2, V,,,., and the K,,, did not change. A primary isotope effect of 1 was found at all pH and temperatures tested and independent of whether H,O or DZO was the solvent. The findings indicate that a step other than the activation of H, was rate-determining in CH=H,MPT+ reduction with H,. This was substantiated by the observation that also the CH=H,MPT+-dependent H,/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. V,,,,, for CH,=H,MPT dehydrogenation to CH-H,MPT+ and H2 was only 2-3 times higher than for CD2=H,MPT dehydrogenation to CD-H,MPT' and HD. Such a small primary isotope effect indicates that the breakage of the C-H bond in the methylene group of CH2=H,MPT was only rate-limiting when hydrogen was substituted by a deuterium.

“…Chaps was purchased from Biomol. Coenzyme F420, coenzyme F430, 5,6,7,8-tetrahydromethanopterin and methanofuran were isolated from M. thermoautotrophicum strain Marburg (Breitung et al, 1992). Coenzyme F410 was purified to apparent homogeneity by HPLC on a LiChrosphere 100 RP-18 column (Borner, 1988).…”

Section: Methodsmentioning

confidence: 99%

H₂: heterodisulfide oxidoreductase complex from Methanobacterium thermoautotrophicum

Setzke

Hedderich

Heiden

et al. 1994

Self Cite

100

The reduction of the heterodisulfide (CoM-S-S-HTP) of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP) with H, is an energy-conserving step in most methanogenic Archaea. In this study, we show that in Methanobacterium thermoautotrophicum (strain Marburg) this reaction is catalyzed by a stable H, :heterodisulfide oxidoreductase complex of F4,,,-non-reducing hydrogenase and heterodisulfide reductase. This complex, which was loosely associated with the cytoplasmic membrane, was purified 17-fold with 80% yield to apparent homogeneity. The purified complex was composed of six different subunits of apparent molecular masses 80, 51, 41, 36, 21 and 17 kDa, and 1 mol complex, with apparent molecular mass 250 kDa, contained approximately 0.6 mol nickel, 0.9 mol FAD, 26 mol non-heme iron and 22 mol acid-labile sulfur. In 25 mM Chaps, the complex partially dissociated into two subcomplexes. The first subcomplex was was composed of the 51-, 41-and 17-kDa subunits; 1 mol trimer contained 0.7 mol nickel, 10 mol non-heme iron and 9 mol acid-labile sulfur and exhibited F,,,-non-reducing hydrogenase activity. The other subcomplex was composed of the 80-, 36-and 21-kDa subunits; 1 mol trimer contained 0.8 mol FAD, 22 mol non-heme iron and 15 mol acid-labile sulfur and exhibited heterodisulfide-reductase activity. The stimulatory effects of potassium phosphate, a membrane component, uracil derivatives and coenzyme F,,, on the H, :heterodisulfide-oxidoreductase activity of the purified complex are described.