David A. Grahame

Methylcobamide:Coenzyme M Methyltransferase Isozymes from Methanosarcina barkeri PHYSICOCHEMICAL CHARACTERIZATION, CLONING, SEQUENCE ANALYSIS, AND HETEROLOGOUS GENE EXPRESSION

A comparative study was made on the physicochemical characteristics of two isozymes of methylcobamide:- coenzyme M methyltransferase (MT2). Both isozymes catalyzed S-methylation of 2-thioethanesulfonate (coenzyme M) and exhibited similar apparent Km values for coenzyme M of 35 μM (MT2-A) and 20 μM (MT2-M). Weak binding to methylcobalamin was indicated by the apparent Km of 14 mM for both isozymes. Cob(I)alamin was established as the major product of the reaction, demonstrating heterolytic cleavage of the methylcobamide carbon-cobalt bond. The isozymes were shown to be zinc-containing metalloproteins. Metal ion chelators strongly inhibited both isozymes. A variety of coenzyme M analogs were tested for activity and/or inhibition. One alternative substrate 3-mercaptopropionate was discovered, with apparent Km 9 mM (MT2-A) and 10 mM (MT2-M). The results suggested an active site geometry in which coenzyme M is bound both by S-coordination to zinc, and electrostatic interaction of the sulfonate with a cationic group on the enzyme. Methanosarcina barkeri genes cmtA and cmtM encoding both isozymes were cloned and sequenced. Both genes encoded proteins with 339 amino acids and predicted molecular masses of 36-37 kDa. Active forms of both isozymes were expressed in Escherichia coli. A conserved segment with the potential for metal binding was found. The possibility of zinc involvement in catalysis of coenzyme M methylation is considered.

Two Separate One-Electron Steps in the Reductive Activation of the A Cluster in Subunit β of the ACDS Complex in Methanosarcina thermophila†

Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex found in methanogens and certain other Archaea that carries out the overall synthesis and cleavage of the acetyl C-C and C-S bonds of acetyl-CoA. The reaction is involved both in the autotrophic fixation of carbon and in the process of methanogenesis from acetate, and takes place at a unique active site metal center known as the A cluster, located on the beta subunit of the ACDS complex and composed of a binuclear Ni-Ni site bridged by a cysteine thiolate to an Fe4S4 center. In this work, a high rate of acetyl-CoA synthesis was achieved with the recombinant ACDS beta subunit by use of methylcobinamide as an appropriate mimic of the physiological base-off corrinoid substrate. The redox dependence of acetyl-CoA synthesis exhibited one-electron Nernst behavior, and the effects of pH on the observed midpoint potential indicated that reductive activation of the A cluster also involves protonation. Initial burst kinetic studies indicated the formation of stoichiometric amounts of an A cluster-acetyl adduct, further supported by direct chromatographic isolation of an active enzyme-acetyl species. Titration experiments indicated that two electrons are required for activation of the enzyme in the process of forming the enzyme-acetyl intermediate. The results also established that the A cluster-acetyl species undergoes reductive elimination of the acetyl group with the simultaneous release of two, low potential electron equivalents. Thus, the one-electron Nernst behavior can be interpreted as the sum of two separate, low potential, one-electron steps. The results tend to exclude reaction mechanisms involving either one- or three-electron reduced forms of the A cluster as immediate precursors to the acetyl species. A scheme involving a [Fe4S4]1+-Ni1+ species is favored over a [Fe4S4]2+-Ni0 form. The role of proton uptake in the possible formation of a Ni2+-hydride intermediate is also discussed. Trapping of electrons during the formation of the A cluster-acetyl species from substrates CO and methylcobinamide was found to be highly favorable, thus presenting a means for extensive activation of the enzyme under otherwise nonpermissive physiological redox potentials.

Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus

Abstract The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction:w CO2 + 2 Fdred(Fe2+) + 2 H+ + CH3– H4SPt + CoA ⇌ acetyl-CoA + H4SPt + 2 Fdox(Fe3+) + H2Owhere Fd is ferredoxin, and CH3–H4SPt and H4SPt denote N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively.

Acetate C–C bond formation and decomposition in the anaerobic world: the structure of a central enzyme and its key active-site metal cluster

The structure of carbon monoxide dehydrogenase/acetyl-coenzyme A synthase (CODH/ACS), a central enzyme in the anaerobic metabolism of acetyl-coenzyme A (acetyl-CoA), has been solved to a resolution of 2.2A. The active-site metal cluster responsible for catalyzing acetyl C-C bond synthesis and cleavage, designated the A center, was identified as an Fe(4)S(4) iron sulfur cluster with one of its cysteine thiolates acting as a bridge to an adjacent binuclear metal site. Nickel was found at one position in the binuclear site and the other metal was indicated to be copper - a surprising result, implying a previously unrecognized role for copper. Details of the A center provided new insight into the unusual organometallic mechanism of acetyl C-C bond formation and cleavage, with substantial conformational changes indicated for binding of the large methylcorrinoid protein substrate, and a unique intramolecular channel acting to contain carbon monoxide within the protein and transfer it to the site needed for acetyl-CoA synthesis.

Tight Coupling of Partial Reactions in the Acetyl-CoA Decarbonylase/Synthase (ACDS) Multienzyme Complex from Methanosarcina thermophila ACETYL C–C BOND FRAGMENTATION AT THE A CLUSTER PROMOTED BY PROTEIN CONFORMATIONAL CHANGES

Direct synthesis and cleavage of acetyl-CoA are carried out by the bifunctional CO dehydrogenase/acetyl-CoA synthase enzyme in anaerobic bacteria and by the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex in Archaea. In both systems, a nickel- and Fe/S-containing active site metal center, the A cluster, catalyzes acetyl C–C bond formation/breakdown. Carbonyl group exchange of [1-14C]acetyl-CoA with unlabeled CO, a hallmark of CODH/ACS, is weakly active in ACDS, and exchange with CO2 was up to 350 times faster, indicating tight coupling of CO release at the A cluster to CO oxidation to CO2 at the C cluster in CO dehydrogenase. The basis for tight coupling was investigated by analysis of three recombinant A cluster proteins, ACDS β subunit from Methanosarcina thermophila, acetyl-CoA synthase of Carboxydothermus hydrogenoformans (ACSCh), and truncated ACSCh lacking its 317-amino acid N-terminal domain. A comparison of acetyl-CoA synthesis kinetics, CO exchange, acetyltransferase, and A cluster Ni+-CO EPR characteristics demonstrated a direct role of the ACS N-terminal domain in promoting acetyl C–C bond fragmentation. Protein conformational changes, related to “open/closed” states previously identified crystallographically, were indicated to have direct effects on the coordination geometry and stability of the A cluster Ni2+-acetyl intermediate, controlling Ni2+-acetyl fragmentation and Ni2+(CO)(CH3) condensation. EPR spectral changes likely reflect variations in the Ni+-CO equatorial coordination environment in closed buried hydrophobic and open solvent-exposed states. The involvement of subunit-subunit interactions in ACDS, versus interdomain contacts in ACS, ensures that CO is not released from the ACDS β subunit in the absence of appropriate interactions with the α2ϵ2 CO dehydrogenase component. The resultant high efficiency CO transfer explains the low rate of CO exchange relative to CO2.

Redox Enzymes of Methanogens: Physicochemical Properties of Selected, Purified Oxidoreductases

The numerous oxidation/reduction reactions involved in the metabolism of methanogens require the participation of equal or greater numbers of different oxidoreductase enzymes. In this chapter only a selected group of these enzymes are considered. A number of additional important redox enzymes including hydrogenase, methylcoenzyme M methylreductase, CoM-S-S-HTP heterodisulfide reductase, dehydrogenases involved in tetrahydromethanopterin-dependent reactions, and formylmethanofuran dehydrogenase are described in other chapters. The purpose of the present chapter is to provide a review with some perspective of the present state of knowledge concerning the structural, catalytic, and physiological functions of a few important oxidoreductase enzymes in methanogens. The examples chosen have been characterized as well-purified preparations and are now among the more extensively studied methanogen oxidoreductase enzymes.

A single operon-encoded form of the acetyl-CoA decarbonylase/synthase multienzyme complex responsible for synthesis and cleavage of acetyl-CoA in Methanosarcina thermophila

Methanogens growing on C-1 substrates synthesize 2-carbon acetyl groups in the form of acetyl-CoA for carbon assimilation using the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) which contains five different subunits encoded within an operon. In species growing on acetate ACDS also functions to cleave the acetate C-C bond for energy production by methanogenesis. A number of species of Methanosarcina that are capable of growth on either C-1 compounds or acetate contain two separate ACDS operons, and questions have been raised about whether or not these operons play separate roles in acetate synthesis and cleavage. Methanosarcina thermophila genomic DNA was analyzed for the presence of two ACDS operons by PCR amplifications with different primer pairs, restriction enzyme analyses, DNA sequencing and Southern blot analyses. A single ACDS operon was identified and characterized, with no evidence for more than one. MALDI mass spectrometric analyses were carried out on ACDS preparations from methanol- and acetate-grown cells. Peptide fragmentation patterns showed that the same ACDS subunits were present regardless of growth conditions. The evidence indicates that a single form of ACDS is used both for acetate cleavage during growth on acetate and for acetate synthesis during growth on C-1 substrates.

Methods for Analysis of Acetyl-CoA Synthase: Applications to Bacterial and Archaeal Systems

The nickel- and iron-containing enzyme acetyl-CoA synthase (ACS) catalyzes de novo synthesis as well as overall cleavage of acetyl-CoA in acetogens, various other anaerobic bacteria, methanogens, and other archaea. The enzyme contains a unique active site metal cluster, designated the A cluster, that consists of a binuclear Ni-Ni center bridged to an [Fe(4)S(4)] cluster. In bacteria, ACS is tightly associated with CO dehydrogenase to form the bifunctional heterotetrameric enzyme CODH/ACS, whereas in archaea, ACS is a component of the large multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS), which comprises five different subunits that make up the subcomponent proteins ACS, CODH, and a corrinoid enzyme. Characteristic properties of ACS are discussed, and key methods are described for analysis of the enzymes multiple redox-dependent activities, including overall acetyl-CoA synthesis, acetyltransferase, and an isotopic exchange reaction between the carbonyl group of acetyl-CoA and CO. Systematic measurement of these activities, applied to different ACS protein forms, provides insight into the ACS catalytic mechanism and physiological functions in both CODH/ACS and ACDS systems.

Different modes of carbon monoxide binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of nickel.

Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO and CH3 units at a unique Ni-Fe cluster, the A cluster, to form an acetyl-Ni intermediate that subsequently reacts with CoA to produce acetyl-CoA. ACS is a component of the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) in Archaea and CO dehydrogenase/ACS (CODH/ACS) in bacteria; in both systems, intraprotein CO channeling takes place between the CODH and ACS active sites. Previous studies indicated that protein conformational changes control the chemical reactivity of the A cluster and suggested the involvement of a conserved Phe residue that moves concomitantly into and out of the coordination environment of Ni. Herein, steady-state rate measurements in which both CO and CH3-corrinoid are varied, and rapid methylation reactions of the ACDS β subunit, measured by stopped-flow methods, provide a kinetic model for acetyl-CoA synthesis that includes a description of the inhibitory effects of CO explained by competition of CO and CH3 for the same form of the enzyme. Electron paramagnetic resonance titrations revealed that the formation of a paramagnetic Ni(+)-CO species does not match the kinetics of CO interaction as a substrate but instead correlates well with an inhibited state of the enzyme, which requires revision of previous models that postulate that this species is an intermediate. Characterization of the β subunit F195A variant showed markedly increased substrate reactivity with CO, which provides biochemical functional evidence of steric shielding of the CO substrate interaction site by the phenyl group side chain. The phenyl group also likely enhances the nucleophilicity of the Ni center to facilitate CH3 group transfer. A model was developed for how the catalytic properties of the A cluster are optimized by linking conformational changes to a repositionable aromatic shield able to modulate the nucleophilicity of Ni, sterically select the most productive order of substrate addition, and overcome intrinsic inhibition by CO.