Characterization of two methanopterin biosynthesis mutants of Methylobacterium extorquens AM1 by use of a tetrahydromethanopterin bioassay

An enzymatic assay was developed to measure tetrahydromethanopterin (H(4)MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1. H(4)MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H(4)MPT biosynthesis. The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5'-phosphate synthase, the first committed step of H(4)MPT biosynthesis. These results provide the first biochemical evidence for H(4)MPT biosynthesis genes in bacteria.     

The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in Methylobacterium extorquens AM1

An NADP-dependent methylene tetrahydromethanopterin (H₄MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO₂ in Methylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the mtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H₄MPT with NADP⁺ rather than with NAD⁺, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H₄F) with NADP⁺. With methylene H₄F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (V_max/K_m) were approximately 20-fold lower than with methylene H₄MPT. Whereas the dehydrogenation of methylene H₄MPT (E₀ = −390 mV) with NADP⁺ (E₀ = −320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H₄F (E₀ = −300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H₄F dehydrogenases from other bacteria and eucarya and with those of methylene H₄MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).     

Implementation of microarrays for Methylobacterium extorquens AM1

Microarrays are an important tool for understanding global gene expression changes, and the resulting data sets can be used to direct physiologic and metabolic studies. To take advantage of this technology, 60-mer oligonucleotide microarrays were designed for Methylobacterium extorquens AM1 to study gene expression changes that occur under differing physiological conditions. The carbon utilization pathways for methanol and succinate have been well characterized, and growth with these substrates was chosen as the condition used to validate the microarray data. The data were analyzed using two different methods and compared to previously obtained experimental data. The array data processed using the Significance Analysis of Microarrays followed by p-value assessment, correlated best to the experimental data. In addition to validating the microarrays, these studies uncovered possible connections between methylotrophy, iron, and sulfur homeostasis, bacteriochlorophyll production and polyketide synthesis, and will likely aid in uncovering further metabolic networks and genes required for methylotrophy.     

The second subunit of methanol dehydrogenase of Methylobacterium extorquens AM1.

The nucleotide and deduced amino acid sequence of a novel small (beta) subunit of methanol dehydrogenase of Methylobacterium extorquens AM1 (previously Pseudomonas AM1) has been determined. Work with the whole protein has shown that is has an alpha 2 beta 2 configuration.     

Arabidopsis mutants with increased sensitivity to aluminum.

Al-sensitive (als) mutants of Arabidopsis were isolated and characterized with the aim of defining mechanisms of Al toxicity and resistance. Most als mutants selected on the basis of root growth sensitivity to Al were recessive, and together the mutants constituted eight complementation groups. Also, in most als mutants, Al sensitivity appeared to be specific for Al relative to La (another trivalent cation), except als2, which was more sensitive to La than wild type. The tendency of roots on mutant seedlings to accumulate Al was examined by staining with morin and hematoxylin, dyes used to indicate the presence of Al. A significant increase in morin staining was observed in als5, consistent with its increased sensitivity to Al. Unexpectedly, als7 and als4 showed less morin staining, suggesting that the roots on these mutants accumulate less Al than wild type seedlings after exposure to Al-containing solutions. Roots of wild-type seedlings produce callose in response to AlCl3 concentrations that inhibit root growth. Only als5 accumulated more callose than wild type in response to low levels (25 mu M) of AICI3 However, als4 and als7 did not accumulate callose at this AlCl3 concentration even though root growth was significantly inhibited. The lack of callose accumulation in als4 and als7 suggests that there is not an obligatory relationship between callose deposition and Al-induced inhibition of root growth.     

The methanol-oxidizing system of Methylobacterium extorquens AM1 reconstituted with purified constituents

The electron transport system (with cytochrome aa3) coupled to the oxidation of methanol in Methylobacterium extorquens AM1 (former Pseudomonas AM1) was reconstituted with highly purified constituents of the system. A mixture of 2.7 microM methanol dehydrogenase, 3.2 microM cytochrome cH, and 71 nM cytochrome c oxidase (= cytochrome aa3) consumed oxygen at a lower rate in the presence of methanol, while its activity was enhanced 3-fold by the addition of 1.4 microM cytochrome cL (74 mol of O2 consumed/mol of heme a of cytochrome c oxidase per min). Further addition of amicyanin to the above mixture did not affect the activity. Although ammonium ion greatly activated the activity of methanol dehydrogenase, the ion had little effect on the oxygen consumption activity of the above mixture. On the basis of the results obtained in the present study, an electron transport system is proposed for the oxidation of methanol in M. extorquens AM1.     

Co-Consumption of Methanol and Succinate by Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 is a facultative methylotrophic Alphaproteobacterium and has been subject to intense study under pure methylotrophic as well as pure heterotrophic growth conditions in the past. Here, we investigated the metabolism of M. extorquens AM1 under mixed substrate conditions, i.e., in the presence of methanol plus succinate. We found that both substrates were co-consumed, and the carbon conversion was two-thirds from succinate and one-third from methanol relative to mol carbon. ¹³C-methanol labeling and liquid chromatography mass spectrometry analyses revealed the different fates of the carbon from the two substrates. Methanol was primarily oxidized to CO₂ for energy generation. However, a portion of the methanol entered biosynthetic reactions via reactions specific to the one-carbon carrier tetrahydrofolate. In contrast, succinate was primarily used to provide precursor metabolites for bulk biomass production. This work opens new perspectives on the role of methylotrophy when substrates are simultaneously available, a situation prevailing under environmental conditions.     

Characterization of the formyltransferase from Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylmethanofuran-tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 microM; apparent Km for H4MPT = 30 microM). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of approximately 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe-4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.     

Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1.

Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+. The purification of a 32-kDa NADP-specific methylene H4MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic alpha-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism. The Km for methylene H4MPT was 50 microM with NAD+ (Vmax = 1100 U x mg(-1) and 100 microM with NADP+ (Vmax = 950 U x mg(-1). The Km value for NAD+ was 200 microM and for NADP+ 20 microM. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal amino-acid 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.     

Glyoxylate regeneration pathway in the methylotroph Methylobacterium extorquens AM1

Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of (14)C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and beta-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.     

Formaldehyde-detoxifying role of the tetrahydromethanopterin-linked pathway in Methylobacterium extorquens AM1

The facultative methylotroph Methylobacterium extorquens AM1 possesses two pterin-dependent pathways for C(1) transfer between formaldehyde and formate, the tetrahydrofolate (H(4)F)-linked pathway and the tetrahydromethanopterin (H(4)MPT)-linked pathway. Both pathways are required for growth on C(1) substrates; however, mutants defective for the H(4)MPT pathway reveal a unique phenotype of being inhibited by methanol during growth on multicarbon compounds such as succinate. It has been previously proposed that this methanol-sensitive phenotype is due to the inability to effectively detoxify formaldehyde produced from methanol. Here we present a comparative physiological characterization of four mutants defective in the H(4)MPT pathway and place them into three different phenotypic classes that are concordant with the biochemical roles of the respective enzymes. We demonstrate that the analogous H(4)F pathway present in M. extorquens AM1 cannot fulfill the formaldehyde detoxification function, while a heterologously expressed pathway linked to glutathione and NAD(+) can successfully substitute for the H(4)MPT pathway. Additionally, null mutants were generated in genes previously thought to be essential, indicating that the H(4)MPT pathway is not absolutely required during growth on multicarbon compounds. These results define the role of the H(4)MPT pathway as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.     

Preliminary crystallographic study of a pseudoazurin from methylotrophic bacterium, Methylobacterium extorquens AM1

Single crystals of pseudoazurin, one of the blue copper proteins produced by methylotrophic bacterium Methylobacterium extorquens AM1, have been obtained by the method of vapor diffusion with ammonium sulfate as a precipitant at pH 8.0. Crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 52.619(4) A, b = 63.280(6) A, c = 35.133(4) A. The asymmetric unit includes one molecule of pseudoazurin (Vm = 2.18 A3/dalton). The crystals are so stable against X-ray irradiation that diffraction intensities of the native crystal up to 1.68 A resolution could be collected from only one crystal. Among the many heavy-metal reagents examined, uranyl acetate gave an effective isomorphous derivative.     

The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: purification and properties.

NAD-dependent formate dehydrogenase (FDH1) was isolated from the alpha-proteobacterium Methylobacterium extorquens AM1 under oxic conditions. The enzyme was found to be a heterodimer of two subunits (alpha1beta1) of 107 and 61 kDa, respectively. The purified enzyme contained per mol enzyme approximately 5 mol nonheme iron and acid-labile sulfur, 0.6 mol noncovalently bound FMN, and approximately 1.8 mol tungsten. The genes encoding the two subunits of FDH1 were identified on the M. extorquens AM1 chromosome next to each other in the order fdh1B, fdh1A. Sequence comparisons revealed that the alpha-subunit harbours putative binding motifs for the molybdopterin cofactor and at least one iron-sulfur cluster. Sequence identity was highest to the catalytic subunits of the tungsten- and selenocysteine-containing formate dehydrogenases characterized from Eubacterium acidaminophilum and Moorella thermoacetica (Clostridium thermoaceticum). The beta-subunit of FDH1 contains putative motifs for binding FMN and NAD, as well as an iron-sulfur cluster binding motif. The beta-subunit appears to be a fusion protein with its N-terminal domain related to NuoE-like subunits and its C-terminal domain related to NuoF-like subunits of known NADH-ubiquinone oxidoreductases.     

Characterization of a novel soluble c-type cytochrome in a moxD mutant of Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 contains a novel c-type cytochrome, called cytochrome c-553, previously thought to be a precursor of the electron acceptor (cytochrome cL) for methanol dehydrogenase. Its amino acid composition and serological characteristics show that it has no structural relationship to cytochrome cL. It usually comprises less than 5% of the total c-type cytochromes. In a moxD mutant, which contains neither methanol dehydrogenase nor cytochrome cL, it comprises 30% of the soluble cytochrome and it has been purified and characterized from that mutant. Cytochrome c-553 is large (Mr 23,000), acidic and monohaem, with a redox potential of 194 mV. It reacts rapidly and completely with CO but is not autoxidizable. It is not autoreducible, and it is not an electron acceptor from methanol dehydrogenase or methylamine dehydrogenase, nor an important electron donor to the oxidase. It is able to accept electrons from cytochrome cL and to donate electrons to cytochrome cH. It is present in the soluble fraction (presumably periplasmic) and membrane fraction of wild-type bacteria during growth on a wide range of growth substrates, but its function in these bacteria or in the moxD mutant has not been determined.     

Biochemical characterization of a dihydromethanopterin reductase involved in tetrahydromethanopterin biosynthesis in Methylobacterium extorquens AM1

During growth on one-carbon (C1) compounds, the aerobic alpha-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H4MPT) derivative dephospho-H4MPT as a C1 carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H4MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H4MPT biosynthesis is the reduction of dihydromethanopterin (H2MPT) to H4MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H2MPT reductase gene (dmrA). In the present work, we describe the biochemical characterization of H2MPT reductase (DmrA), which is encoded by dmrA. The gene was expressed with a six-histidine tag in Escherichia coli, and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H2MPT with a specific activity of 2.8 micromol of NADPH oxidized per min per mg of protein at 30 degrees C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H2MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F420, used, respectively, in bacteria and methanogenic archaea.     

Nucleotide sequence of the amicyanin gene from Methylobacterium extorquens AM1.

The gene for amicyanin from the methylotrophic bacterium, Methylobacterium extorquens AM1 was identified. It encodes a protein consisting of 119 amino acids with a molecular weight of 12,609 kDa. The amino acid sequence shows the presence of a typical leader sequence and signal peptidase recognition site. Two putative hairpin structures were found, one located directly behind the amicyanin gene and another located 50 bp upstream. The same sequence AAAATCCC was found near the start codons for the small subunit of methylamine dehydrogenase and amicyanin, but its significance is not known.     

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1.

An isolated 5.2-kb fragment of Methylobacterium extorquens AM1 DNA was found to contain a gene cluster involved in methylamine utilization. Analysis of polypeptides synthesized in an Escherichia coli T7 expression system showed that five genes were present. Two of the genes encoded the large and small subunits of methylamine dehydrogenase, and a third encoded amicyanin, the presumed electron acceptor for methylamine dehydrogenase, but the function of the other two genes is not known. The order on the 5.2-kb fragment was found to be large-subunit gene, the two genes of unknown function, small-subunit gene, amicyanin gene. The gene for azurin, another possible electron acceptor in methylamine oxidation, does not appear to be present within this cluster of methylamine utilization genes.