Four families of folate-independent methionine synthases

Although most organisms synthesize methionine from homocysteine and methyl folates, some have “core” methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.     

On the origin of biochemistry at an alkaline hydrothermal vent

A model for the origin of biochemistry at an alkaline hydrothermal vent has been developed that focuses on the acetyl-CoA (Wood-Ljungdahl) pathway of CO2 fixation and central intermediary metabolism leading to the synthesis of the constituents of purines and pyrimidines. The idea that acetogenesis and methanogenesis were the ancestral forms of energy metabolism among the first free-living eubacteria and archaebacteria, respectively, stands in the foreground. The synthesis of formyl pterins, which are essential intermediates of the Wood-Ljungdahl pathway and purine biosynthesis, is found to confront early metabolic systems with steep bioenergetic demands that would appear to link some, but not all, steps of CO2 reduction to geochemical processes in or on the Earth's crust. Inorganically catalysed prebiotic analogues of the core biochemical reactions involved in pterin-dependent methyl synthesis of the modern acetyl-CoA pathway are considered. The following compounds appear as probable candidates for central involvement in prebiotic chemistry: metal sulphides, formate, carbon monoxide, methyl sulphide, acetate, formyl phosphate, carboxy phosphate, carbamate, carbamoyl phosphate, acetyl thioesters, acetyl phosphate, possibly carbonyl sulphide and eventually pterins. Carbon might have entered early metabolism via reactions hardly different from those in the modern Wood-Ljungdahl pathway, the pyruvate synthase reaction and the incomplete reverse citric acid cycle. The key energy-rich intermediates were perhaps acetyl thioesters, with acetyl phosphate possibly serving as the universal metabolic energy currency prior to the origin of genes. Nitrogen might have entered metabolism as geochemical NH3 via two routes: the synthesis of carbamoyl phosphate and reductive transaminations of alpha-keto acids. Together with intermediates of methyl synthesis, these two routes of nitrogen assimilation would directly supply all intermediates of modern purine and pyrimidine biosynthesis. Thermodynamic considerations related to formyl pterin synthesis suggest that the ability to harness a naturally pre-existing proton gradient at the vent-ocean interface via an ATPase is older than the ability to generate a proton gradient with chemistry that is specified by genes.     

Rapid kinetic studies of acetyl-CoA synthesis: evidence supporting the catalytic intermediacy of a paramagnetic NiFeC species in the autotrophic Wood-Ljungdahl pathway

CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), a key enzyme in the Wood-Ljungdahl pathway of anaerobic CO(2) fixation, is a bifunctional enzyme containing CODH, which catalyzes the reversible two-electron oxidation of CO to CO(2), and ACS, which catalyzes acetyl-CoA synthesis from CoA, CO, and a methylated corrinoid iron-sulfur protein (CFeSP). ACS contains an active site nickel iron-sulfur cluster that forms a paramagnetic adduct with CO, called the nickel iron carbon (NiFeC) species, which we have hypothesized to be a key intermediate in acetyl-CoA synthesis. This hypothesis has been controversial. Here we report the results of steady-state kinetic experiments; stopped-flow and rapid freeze-quench transient kinetic studies; and kinetic simulations that directly test this hypothesis. Our results show that formation of the NiFeC intermediate occurs at approximately the same rate as, and its decay occurs 6-fold faster than, the rate of acetyl-CoA synthesis. Kinetic simulations of the steady-state and transient kinetic results accommodate the NiFeC species in the mechanism and define the rate constants for the elementary steps in acetyl-CoA synthesis. The combined results strongly support the kinetic competence of the NiFeC species in the Wood-Ljungdahl pathway. The results also imply that the methylation of ACS occurs by attack of the Ni(1+) site in the NiFeC intermediate on the methyl group of the methylated CFeSP. Our results indicate that CO inhibits acetyl-CoA synthesis by inhibiting this methyl transfer reaction. Under noninhibitory CO concentrations (below 100 microM), formation of the NiFeC species is rate-limiting, while at higher inhibitory CO concentrations, methyl transfer to ACS becomes rate-limiting.     

The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum)

This paper describes the genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the Wood-Ljungdahl pathway of CO and CO(2) fixation. This pathway, which is also known as the reductive acetyl-CoA pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into 3 mol of acetate and to grow autotrophically using H(2) and CO as electron donors and CO(2) as an electron acceptor. Methanogenic archaea use this pathway in reverse to grow by converting acetate into methane and CO(2). Acetogenic bacteria also couple the Wood-Ljungdahl pathway to a variety of other pathways to allow the metabolism of a wide variety of carbon sources and electron donors (sugars, carboxylic acids, alcohols and aromatic compounds) and electron acceptors (CO(2), nitrate, nitrite, thiosulfate, dimethylsulfoxide and aromatic carboxyl groups). The genome consists of a single circular 2 628 784 bp chromosome encoding 2615 open reading frames (ORFs), which includes 2523 predicted protein-encoding genes. Of these, 1834 genes (70.13%) have been assigned tentative functions, 665 (25.43%) matched genes of unknown function, and the remaining 24 (0.92%) had no database match. A total of 2384 (91.17%) of the ORFs in the M. thermoacetica genome can be grouped in orthologue clusters. This first genome sequence of an acetogenic bacterium provides important information related to how acetogens engage their extreme metabolic diversity by switching among different carbon substrates and electron donors/acceptors and how they conserve energy by anaerobic respiration. Our genome analysis indicates that the key genetic trait for homoacetogenesis is the core acs gene cluster of the Wood-Ljungdahl pathway.     

The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein

This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an SN2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center.     

Evidence for the presence of a CmuA methyltransferase pathway in novel marine methyl halide-oxidizing bacteria

Marine bacteria that oxidized methyl bromide and methyl chloride were enriched and isolated from seawater samples. Six methyl halide-oxidizing enrichments were established from which 13 isolates that grew on methyl bromide and methyl chloride as sole sources of carbon and energy were isolated and maintained. All isolates belonged to three different clades in the Roseobacter group of the alpha subdivision of the Proteobacteria and were distinct from Leisingera methylohalidivorans, the only other identified marine bacterium that grows on methyl bromide as sole source of carbon and energy. Genes encoding the methyltransferase/corrinoid-binding protein CmuA, which is responsible for the initial step of methyl chloride oxidation in terrestrial methyl halide-oxidizing bacteria, were detected in enrichments and some of the novel marine strains. Gene clusters containing cmuA and other genes implicated in the metabolism of methyl halides were cloned from two of the isolates. Expression of CmuA during growth on methyl halides was demonstrated by analysis of polypeptides expressed during growth on methyl halides by SDS-PAGE and mass spectrometry in two isolates representing two of the three clades. These findings indicate that certain marine methyl halide degrading bacteria from the Roseobacter group contain a methyltransferase pathway for oxidation of methyl bromide that may be similar to that responsible for methyl chloride oxidation in Methylobacterium chloromethanicum. This pathway therefore potentially contributes to cycling of methyl halides in both terrestrial and marine environments.     

Interaction of PvALF and VP1 B3 domains with the β-phaseolin promoter

Caffeine (1,3,7-trimethylxanthine) is derived from xanthosine through three successive transfers of methyl groups and a single ribose removal in coffee plants. The methyl group transfer is catalyzed by N-zmethyltransferases, xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT). We previously cloned three genes encoding each of these N-methyltransferases from coffee plants, and reconstituted the final sequence of the caffeine synthetic pathway in vitro. In the present study, we simultaneously expressed these coffee genes in tobacco plants (Nicotiana tabacum), using a multiple-gene transfer method, and confirmed successful caffeine production up to 5 μg g⁻¹ fresh weight in leaves of the resulting transgenic plants. Their effects on feeding behavior of tobacco cutworms (Spodoptera litura), which damage a wide range of crops, were then examined. Leaf disc choice test showed that caterpillars selectively fed on the wild-type control materials, or positively avoided the transgenic materials. The results suggest a novel approach to confer self-defense by producing caffeine in planta. A second generation of transgenic crops containing caffeine may save labor and agricultural costs and also mitigate the environmental load of pesticides in future.     

Pulse-chase studies of the synthesis of acetyl-CoA by carbon monoxide dehydrogenase/acetyl-CoA synthase: evidence for a random mechanism of methyl and carbonyl addition

Carbon monoxide dehydrogenase/acetyl-CoA synthase catalyzes acetyl-CoA synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein, which acts as a methyl donor. This reaction is the last step in the Wood-Ljungdahl pathway of anaerobic carbon fixation. The binding sequence for the three substrates has been debated for over a decade. Different binding orders imply different mechanisms (i.e. paramagnetic versus diamagnetic mechanisms). Ambiguity arises because CO and CoA can each undergo isotopic exchange with acetyl-CoA, suggesting that either of these two substrates could be the last to bind to the acetyl-CoA synthase active site. Furthermore, carbonylation, CoA binding, and methyl transfer can all occur in the absence of the other two substrates. Here, we report pulse-chase studies, which unambiguously establish the order in which the three substrates bind. Although a CoA pulse is substantially diluted by excess CoA in the chase, isotope recovery of a pulse of labeled CO or methyl group is unaffected by the presence of excess unlabeled CO or methyl group in the chase. These results demonstrate that CoA is the last substrate to bind and that CO and the methyl group bind randomly as the first substrate in acetyl-CoA synthesis. Up to 100% of the methyl groups and CoA and up to 60-70% of the CO employed in the pulse phase can be trapped in the product acetyl-CoA.     

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.     

Identification and characterization of oxalate oxidoreductase, a novel thiamine pyrophosphate-dependent 2-oxoacid oxidoreductase that enables anaerobic growth on oxalate

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 μM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.     

The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases.     

Structural basis for electron and methyl-group transfer in a methyltransferase system operating in the reductive acetyl-CoA pathway

Several anaerobic acetogenic, methanogenic, hydrogenogenic, and sulfate-reducing microorganisms are able to use the reductive acetyl-CoA (Wood-Ljungdahl) pathway to convert CO₂ into biomass. The reductive acetyl-CoA pathway consists of two branches connected by the Co/Fe-containing corrinoid iron-sulfur protein (CoFeSP), which transfers a methyl group from a methyltransferase (MeTr)/methyltetrahydrofolate (CH₃-H₄ folate) complex to the reduced Ni-Ni-[4Fe-4S] cluster (cluster A) of acetyl-CoA synthase. We investigated the CoFeSP and MeTr couple of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans and show that the two proteins are able to catalyze the methyl-group transfer reaction from CH₃-H₄ folate to the Co(I) center of CoFeSP. We determined the crystal structures of both proteins. The structure of CoFeSP includes the previously unresolved N-terminal domain of the large subunit of CoFeSP, revealing a unique four-helix-bundle-like architecture in which a [4Fe-4S] cluster is shielded by hydrophobic amino acids. It further reveals that the corrinoid and the [4Fe-4S] cluster binding domains are mobile, which is mandatory for the postulated electron transfer between them. Furthermore, we solved the crystal structures of apo-MeTr, CH₃-H₄-folate-bound MeTr, and H₄-folate-bound MeTr, revealing a substrate-induced closure of the CH₃-H₄ folate binding cavity of MeTr. We observed three different conformations of Asn200 depending on the substrate bound in the active site, demonstrating its conformational modulation by hydrogen-bonding interactions with the substrate. The observed flexibility could be essential to stabilize the transition state during methyl-group transfer. The conformational space and role of Asn200 are likely conserved in homologous cobalamin-dependent MeTrs such as methionine synthase.     

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.     

Biosynthesis and emission of insect-induced methyl salicylate and methyl benzoate from rice

Two benzenoid esters, methyl salicylate (MeSA) and methyl benzoate (MeBA), were detected from insect-damaged rice plants. By correlating metabolite production with gene expression analysis, five candidate genes encoding putative carboxyl methyltransferases were identified. Enzymatic assays with Escherichia coli-expressed recombinant proteins demonstrated that only one of the five candidates, OsBSMT1, has salicylic acid (SA) methyltransferase (SAMT) and benzoic acid (BA) methyltransferase (BAMT) activities for producing MeSA and MeBA, respectively. Whereas OsBSMT1 is phylogenetically relatively distant from dicot SAMTs, the three-dimensional structure of OsBSMT1, which was determined using homology-based structural modeling, is highly similar to those of characterized SAMTs. Analyses of OsBSMT1 expression in wild-type rice plants under various stress conditions indicate that the jasmonic acid (JA) signaling pathway plays a critical role in regulating the production and emission of MeSA in rice. Further analysis using transgenic rice plants overexpressing NH1, a key component of the SA signaling pathway in rice, suggests that the SA signaling pathway also plays an important role in governing OsBSMT1 expression and emission of its products, probably through a crosstalk with the JA signaling pathway. The role of the volatile products of OsBSMT1, MeSA and MeBA, in rice defense against insect herbivory is discussed.