Methyl Sulfide Production by a Novel Carbon Monoxide Metabolism in Methanosarcina acetivorans

We observed dimethyl sulfide and methanthiol production in pure incubations of the methanogen Methanosarcina acetivorans when carbon monoxide (CO) served as the only electron donor. Energy conservation likely uses sodium ion gradients for ATP synthesis. This novel metabolism permits utilization of CO by the methanogen, resulting in quantitative sulfide methylation.     

Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans

Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated Macs_Ma, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PP_i were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PP_i. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The Macs_Ma structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys²⁵⁶, Cys²⁹⁸, Gly³⁵¹, Trp²⁵⁹, Trp²³⁷, and Trp²⁵⁴, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.AMP-forming acetyl coenzyme A (acetyl-CoA) synthetase (Acs; acetate:CoA ligase [AMP forming], EC 6.2.1.1), which catalyzes the activation of acetate to acetyl-CoA, is a member of the acyl-adenylate-forming enzyme superfamily (8), which consists of acyl- and aryl-CoA ligases, nonribosomal peptide synthetases that mediate the synthesis of peptide and polyketide secondary metabolites, such as gramicidin and tyrocidine, and the enzymes firefly luciferase and α-aminoadipate reductase. Although these enzymes share the property of forming an acyl-adenylate intermediate and are structurally related, they share limited sequence homology and catalyze unrelated reactions in which the intermediate serves different functions for different members of this enzyme family.A two-step mechanism for Acs (equations 1 and 2) in which the reaction proceeds through an acetyl-AMP intermediate has been proposed based on evidence including detection of an enzyme-bound acetyl-AMP (2-4, 38): (1) (2)In the CoA-dependent first step of the reaction, an enzyme-bound acetyl-AMP intermediate is formed from acetate and ATP, and inorganic pyrophosphate (PP_i) is released. In the second step, the acetyl group is transferred to the sulfhydryl group of CoA and AMP is released. Other short- (Sacs) and medium-chain acyl-CoA synthetases (Macs) follow a similar reaction mechanism using acyl substrates other than acetate (8, 15).In the 2.3-Å structure of trimeric Saccharomyces cerevisiae Acs1 in a binary complex with AMP (19), the C-terminal domain is positioned away from the N-terminal domain in a conformation for catalysis of the first step of the reaction (equation 1). The 1.75-Å structure of the monomeric Salmonella enterica Acs (Acs_Se) (13) in complex with both CoA and adenosine-5′-propylphosphate, an inhibitor of the related propionyl-CoA synthetase (12, 15), which mimics the acetyl-adenylate intermediate, reveals that the C-terminal domain of Acs is rotated approximately 140° toward the N-terminal domain to form the complete active site for catalysis of the second half-reaction (equation 2). In this orientation, the CoA thiol is properly positioned for nucleophilic attack on the acetyl group. In structure/function studies of 4-chlorobenzoate:CoA ligase (CBAL), a distant member of the acyl- and aryl-CoA synthetase subfamily of the acyl-adenylate-forming enzyme superfamily, Wu et al. (39) and Reger et al. (28) provide evidence that PP_i produced in the first step of the reaction dissociates from the enzyme before the switch from the first conformation to the second conformation required for CoA binding and catalysis of the second step of the reaction.Acs and Sacs/Macs are widespread in all three domains of life and play a key role in archaea, as suggested by the finding that several thermophilic archaea have multiple open reading frames (ORFs) (up to seven) that encode putative Sacs or Macs (33). The chemolithoautotrophic methanoarchaeon Methanothermobacter thermautotrophicus has two ORFs with high identity to Acs and a third ORF that is likely to encode a Macs. M. thermautotrophicus Acs1 (Acs1_Mt) has been biochemically and kinetically characterized, has been shown to have a strong preference for acetate as the acyl substrate, and can also utilize propionate but not butyrate (16, 17).Methanosarcina and Methanosaeta are the only two methanoarchaea isolated that are able to utilize acetate as substrate for methane production. Unlike Methanosaeta species, which utilize Acs for catalyzing the first step of methanogenesis (18, 34), Methanosarcina species employ the acetate kinase-phosphotransacetylase pathway for activation of acetate to acetyl-CoA, and an Acs activity has not been observed in Methanosarcina (1, 23, 30, 32). Surprisingly, an Acs-related sequence was identified in the Methanosarcina acetivorans genome. Here we describe the kinetic characterization this enzyme, designated Macs_Ma, and show that it utilizes longer acyl substrates than Acs. The preferred acyl substrate was shown to be 2-methylbutyrate, and 2-methylbutyryl-CoA, AMP, and PP_i were the products of the reaction, as expected. However, when propionate was used as the acyl substrate, propionyl-CoA was not produced. Instead, in the absence of CoA, propionyl-AMP and PP_i were released, whereas in the presence of CoA, the propionyl-AMP intermediate was broken down into AMP and propionate and released along with PP_i. Intermediate results were obtained with other acyl substrates, with both acyl-CoA and acyl-AMP production observed.The 2.1-Å crystal structure of Macs_Ma (31), determined in the absence of any substrate, revealed the enzyme to be in a conformation similar to that of the S. enterica Acs (13) with respect to the position of the C-terminal domain. Through inspection of the Macs_Ma structure and alignment of Acs, Sacs, and Macs sequences, we identified six residues that form the putative acyl substrate-binding pocket. Individual alterations at these residues dramatically diminished enzyme activity and indicate that the acyl substrate-binding pocket of Macs_Ma has a very precise architecture that cannot be perturbed.     

Negative-Stain Electron Microscopy Reveals Dramatic Structural Rearrangements in Ni-Fe-S-Dependent Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase

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Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, is a dimeric haem-protein whose biological role is still unknown. As other globins, protoglobin can bind O₂, CO and NO reversibly in vitro, but it displays specific functional and structural properties within members of the hemoglobin superfamily. CO binding to and dissociation from the haem occurs through biphasic kinetics, which arise from binding to (and dissociation from) two distinct tertiary states in a ligation-dependent equilibrium. From the structural viewpoint, protoglobin-specific loops and a N-terminal extension of 20 residues completely bury the haem within the protein matrix. Thus, access of small ligand molecules to the haem is granted by two apolar tunnels, not common to other globins, which reach the haem distal site from locations at the B/G and B/E helix interfaces. Here, the roles played by residues Trp(60)B9, Tyr(61)B10 and Phe(93)E11 in ligand recognition and stabilization are analyzed, through crystallographic investigations on the ferric protein and on selected mutants. Specifically, protein structures are reported for protoglobin complexes with cyanide, with azide (also in the presence of Xenon), and with more bulky ligands, such as imidazole and nicotinamide. Values of the rate constant for cyanide dissociation from ferric MaPgb-cyanide complexes have been correlated to hydrogen bonds provided by Trp(60)B9 and Tyr(61)B10 that stabilize the haem-Fe(III)-bound cyanide. We show that protoglobin can strikingly reshape, in a ligand-dependent way, the haem distal site, where Phe(93)E11 acts as ligand sensor and controls accessibility to the haem through the tunnel system by modifying the conformation of Trp(60)B9.     

A Heme-based Redox Sensor in the Methanogenic Archaeon Methanosarcina acetivorans

Based on a bioinformatics study, the protein MA4561 from the methanogenic archaeon Methanosarcina acetivorans was originally predicted to be a multidomain phytochrome-like photosensory kinase possibly binding open-chain tetrapyrroles. Although we were able to show that recombinantly produced and purified protein does not bind any known phytochrome chromophores, UV-visible spectroscopy revealed the presence of a heme tetrapyrrole cofactor. In contrast to many other known cytoplasmic heme-containing proteins, the heme was covalently attached via one vinyl side chain to cysteine 656 in the second GAF domain. This GAF domain by itself is sufficient for covalent attachment. Resonance Raman and magnetic circular dichroism data support a model of a six-coordinate heme species with additional features of a five-coordination structure. The heme cofactor is redox-active and able to coordinate various ligands like imidazole, dimethyl sulfide, and carbon monoxide depending on the redox state. Interestingly, the redox state of the heme cofactor has a substantial influence on autophosphorylation activity. Although reduced protein does not autophosphorylate, oxidized protein gives a strong autophosphorylation signal independent from bound external ligands. Based on its genomic localization, MA4561 is most likely a sensor kinase of a two-component system effecting regulation of the Mts system, a set of three homologous corrinoid/methyltransferase fusion protein isoforms involved in methyl sulfide metabolism. Consistent with this prediction, an M. acetivorans mutant devoid of MA4561 constitutively synthesized MtsF. On the basis of our results, we postulate a heme-based redox/dimethyl sulfide sensory function of MA4561 and propose to designate it MsmS (methyl sulfide methyltransferase-associated sensor).     

Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO₂ ^– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are k _app1 = 9.6±0.2 M^–1 s^–1 and k _app2 = 1.2±0.1 M^–1 s^–1 (at pH 7.4 and 20°C). The k _app1 and k _app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h _app = 3.8×10⁴ M^–1 s^–1 and h ₀ = 2.8×10^–1 s^–1 (at pH 7.4 and 20°C). The pH-dependence of h _on and h ₀ values reflects the acid-base equilibrium of peroxynitrite (pK _a = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.     

Influence of carbon monoxide on metabolite formation in Methanosarcina acetivorans

Methanogenic archaea conserve energy for growth by reducing some one- and two-carbon compounds to methane and concomitantly generating an ion motive force. Growth of Methanosarcina acetivorans on carbon monoxide (CO) is peculiar as it involves formation of, besides methane, formate, acetate and methylated thiols. It has been argued that methane formation is partially inhibited under carboxidotrophic conditions and that the other products result from either detoxification of CO or from bypassing methanogenesis with other pathways for energy conservation. To gain a deeper understanding of the CO-dependent physiology of M. acetivorans we analyzed metabolite formation in resting cells. The initial rates of methane, acetate, formate, and dimethylsulfide formation increased differentially with increasing CO concentrations but were maximal already at the same moderate CO partial pressure. Strikingly, further increase of the amount of CO was not inhibitory. The maximal rate of methane formation from CO was approximately fivefold lower than that from methanol, consistent with the previously observed significant downregulation of the energy converting sodium-dependent methyltransferase. The rate of dimethylsulfide formation from CO was only 1–2% of that of methane formation under any conditions tested. Implications of the data presented for previously proposed pathways of CO utilization are discussed.     

Differential regulation of the three methanol methyltransferase isozymes in Methanosarcina acetivorans C2A

Genetic analysis of the three methanol-specific methyltransferase 1 operons (mtaCB1, mtaCB2, and mtaCB3) in Methanosarcina acetivorans led to the suggestion that each of them has a discrete function during growth on methanol, which might be reflected in differential gene regulation (Pritchett and Metcalf, Mol. Microbiol. 56:1183-1194, 2005). To test this suggestion, reporter gene fusions were constructed for each of the three operons, and their expression was examined under various growth conditions. Expression of the mtaCB1 and mtaCB2 fusions was 100-fold and 575-fold higher, respectively, in methanol-grown cells than in trimethylamine (TMA)-grown cells. The mtaCB3 fusion was expressed at low levels on methanol, TMA, and dimethylamine but was significantly upregulated on monomethylamine and acetate. When TMA- or acetate-grown cultures were shifted to methanol, the mtaCB1 fusion was expressed most highly during exponential phase, whereas the mtaCB2 fusion, although strongly induced prior to mtaCB1 expression, did not reach full expression levels until stationary phase. The mtaCB3 fusion was transiently expressed prior to entry into exponential phase during a TMA-to-methanol substrate shift experiment. When acetate-grown cells were shifted to medium containing both TMA and methanol, TMA utilization commenced prior to utilization of methanol; however, these two substrates were consumed simultaneously later in growth. Under these conditions expression of the mtaCB2 and mtaCB3 fusions was delayed, suggesting that methylamines may repress their expression.     

The Hydrogenase Activity of the Molybdenum/Copper-containing Carbon Monoxide Dehydrogenase of Oligotropha carboxidovorans

The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H₂ has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H₂ with a limiting rate constant of 5.3 s⁻¹ and a dissociation constant K_d of 525 μm; both k_red and k_red/K_d, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H₂, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site ^63,65Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H₂ oxidation is proposed which involves initial binding of H₂ to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.     

In vivo role of three fused corrinoid/methyl transfer proteins in Methanosarcina acetivorans

Methanosarcina acetivorans is able to use carbon monoxide (CO) as the sole source of energy for growth. Its carboxidotrophic growth is peculiar as it involves formation of acetate, formate and methylated thiols, besides methane. Under this condition three proteins homologous to both corrinoid proteins and methyltransferases (MA0859, MA4384 and MA4558) are highly abundant. To address their role in M. acetivorans , a set of single and double mutants, and the triple mutant, was constructed by deletion/disruption of the encoding genes. Phenotypic analysis of the mutants rules out an important role of the methyltransferase homologues in the CO₂ reduction pathway of methanogenesis. Instead, the single and double mutants were affected to various degrees in their capacity to generate dimethylsulphide (DMS) from CO and to form methane from DMS. The triple mutant was unable to produce or metabolize DMS, and could not grow with DMS as the sole energy source, which demonstrates that MA0859, MA4384 and MA4558 are involved in, and required for, methylsulphide metabolism of M. acetivorans . Based on these findings we propose to designate MA0859, MA4384 and MA4558 as m ethyl t ransferases specific for methyl s ulphides, MtsD, MtsF and MtsH respectively.     

Structural heterogeneity and ligand gating in ferric Methanosarcina acetivorans protoglobin mutants

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, displays peculiar structural and functional properties within members of the hemoglobin superfamily. In fact, MaPgb-specific loops and a N-terminal extension (20 amino acid residues) completely bury the heme within the protein matrix. Therefore, the access of diatomic gaseous molecules to the heme is granted by two apolar tunnels reaching the heme distal site from locations at the B/G and B/E helix interfaces. The presence of two tunnels within the protein matrix could be partly responsible for the slightly biphasic ligand binding behavior. Unusually, MaPgb oxygenation is favored with respect to carbonylation. Here, the crucial role of Tyr(B10)61 and Ile(G11)149 residues, located in the heme distal site and lining the protein matrix tunnels 1 and 2, respectively, on ligand binding to the heme-Fe-atom and on distal site structural organization is reported. In particular, tunnel 1 accessibility is modulated by a complex reorganization of the Trp(B9)60 and Phe(E11)93 side-chains, triggered by mutations of the Tyr(B10)61 and Ile(G11)149 residues, and affected by the presence and type of the distal heme-bound ligand.     

Adrenergic Regulation of the Reduction in Acetyl Coenzyme A: Arylamine 7V-Acetyltransferase Activity in the Rat Pineal

Abstract: Pharmacologically active agents were employed to study the mechanisms that control the reduction in levels of acetyl-coA: arylamine N-acetyltransferase activity (NAT) (EC 2.3.1.5) in the rat pineal. Pretreatment of rats with phenoxybenzamine or phentolamine prevented the rapid light-mediated decrease in NAT activity, although pretreatment with yohimbine or atropine did not alter this effect of light. Administration of mecamylamine resulted in a rapid reduction in enzyme activity prior to light exposure. When clonidine was administered intraperitoneally to animals with elevated NAT levels, there was a rapid decrease in enzyme activity, mimicking the effects of light. However, intraperitoneal injections of norepinephrine, methoxamine and phenylephrine into similar groups of animals had no significant effect on enzyme acitivity. When clonidine and norepinephrine were administered intraventricularly, there was a rapid reduction in enzyme activity. On the other hand, intraventricular administration of phenylephrine did not result in reduced enzyme activity. Pretreatment of animals with phenoxybenzamine failed to block the reduction in NAT activity precipitated by low doses of clonidine. This clonidine-mediated reduction in enzyme activity was, however, blocked by yohimbine. When animals were simultaneously exposed to light and administered clonidine, the rapid reduction in NAT activity was affected only when animals were pretreated with both yohimbine and phenoxybenzamine. In contrast to the decrease in pineal NAT activity observed in in vivo preparations, incubation of pineals with clonidine in an organ culture system produced a moderate, but consistent, rise in enzyme activity. These results suggest that stimulation of a receptor with α-adrenergic characteristics mediates the reduction in NAT activity produced by light. Stimulation of yet a second adrenergic-like receptor appears to mediate a reduction in pineal NAT activity precipitated by clonidine. Our evidence suggests that one or both of these receptors are located within the central nervous system.     

Structural and biochemical characterization of flavoredoxin from the archaeon Methanosarcina acetivorans

Flavoredoxin is a FMN-containing electron transfer protein that functions in the energy-yielding metabolism of Desulfovibrio gigas of the Bacteria domain. Although characterization of this flavoredoxin is the only one reported, a database search revealed homologues widely distributed in both the Bacteria and Archaea domains that define a novel family. To improve our understanding of this family, a flavoredoxin from Methanosarcina acetivorans of the Archaea domain was produced in Escherichia coli and biochemically characterized, and a high-resolution crystal structure was determined. The protein was shown to be a homodimer with a subunit molecular mass of 21 kDa containing one noncovalently bound FMN per monomer. Redox titration showed an E(m) of -271 mV with two electrons, consistent with no semiquinone observed in the potential range studied, a result suggesting the flavoredoxin functions as a two-electron carrier. However, neither of the obligate two-electron carriers, NAD(P)H and coenzyme F420H2, was a competent electron donor, whereas 2[4Fe-4S] ferredoxin reduced the flavoredoxin. The X-ray crystal structure determined at 2.05 A resolution revealed a homodimer containing one FMN per monomer, consistent with the biochemical characterization. The isoalloxazine ring of FMN was shown buried within a narrow groove approximately 10 A from the positively charged protein surface that possibly facilitates interaction with the negatively charged ferredoxin. The structure provides a basis for predicting the mechanism by which electrons are transferred between ferredoxin and FMN. The FMN is bound with hydrogen bonds to the isoalloxazine ring and electrostatic interactions with the phosphate moiety that, together with sequence analyses of homologues, indicate a novel FMN binding motif for the flavoredoxin family.