Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum.

Clostridium autoethanogenum, the bacterial model for biological conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concentrations. The strictly anaerobic bacterium derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient molecular machineries for CO-conversion. Here, we structurally and biochemically characterized the key enzyme of the CO-converting metabolism: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2ß2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystallization artefact. Therefore, the internal CO-channeling system, critical to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidation and acetyl-CoA production. Biochemical experiments showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidation and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.

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

The Ni-Fe-S-containing A-cluster of acetyl-coenzyme A (CoA) synthase (ACS) assembles acetyl-CoA from carbon monoxide (CO), a methyl group (CH3+), and CoA. To accomplish this feat, ACS must bind CoA and interact with two other proteins that contribute the CO and CH3+, respectively: CO dehydrogenase (CODH) and corrinoid Fe-S protein (CFeSP). Previous structural data show that, in the model acetogen Moorella thermoacetica, domain 1 of ACS binds to CODH such that a 70-Å-long internal channel is created that allows CO to travel from CODH to the A-cluster. The A-cluster is largely buried and is inaccessible to CFeSP for methylation. Here we use electron microscopy to capture multiple snapshots of ACS that reveal previously uncharacterized domain motion, forming extended and hyperextended structural states. In these structural states, the A-cluster is accessible for methylation by CFeSP.

Insights into the Chemical Reactivity in Acetyl-CoA Synthase.

The biological synthesis of acetyl-coenzyme A (acetyl-CoA), catalyzed by acetyl-CoA synthase (ACS), is of biological significance and chemical interest acting as a source of energy and carbon. The catalyst contains an unusual hexa-metal cluster with two nickel ions and a [Fe4S4] cluster. DFT calculations have been performed to investigate the ACS reaction mechanism starting from three different oxidation states (+2, +1, and 0) of Nip, the nickel proximal to [Fe4S4]. The results indicate that the ACS reaction proceeds first through a methyl radical transfer from cobalamin (Cbl) to Nip randomly accompanying with the CO binding. After that, C-C bond formation occurs between the Nip-bound methyl and CO, forming Nip-acetyl. The substrate CoA-S- then binds to Nip, allowing C-S bond formation between the Nip-bound acetyl and CoA-S-. Methyl transfer is rate-limiting with a barrier of ∼14 kcal/mol, which does not depend on the presence or absence of CO. Both the Nip2+ and Nip1+ states are chemically capable of catalyzing the ACS reaction independent of the state (+2 or +1) of the [Fe4S4] cluster. The [Fe4S4] cluster is not found to affect the steps of methyl transfer and C-C bond formation but may be involved in the C-S bond formation depending on the detailed mechanism chosen. An ACS active site containing a Nip(0) state could not be obtained. Optimizations always led to a Nip1+ state coupled with [Fe4S4]1+. The calculations show a comparable activity for Nip1+/[Fe4S4]1+, Nip1+/[Fe4S4]2+, and Nip2+/[Fe4S4]2+. The results here give significant insights into the chemistry of the important ACS reaction.

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.

Crystal structures of Moorella thermoacetica cyanuric acid hydrolase reveal conformational flexibility and asymmetry important for catalysis.

An ancient enzyme family responsible for the catabolism of the prebiotic chemical cyanuric acid (1,3,5-triazine-2,4,6-triol) was recently discovered and is undergoing proliferation in the modern world due to industrial synthesis and dissemination of 1,3,5-triazine compounds. Cyanuric acid has a highly stabilized ring system such that bacteria require a unique enzyme with a novel fold and subtle active site construction to open the ring. Each cyanuric acid hydrolase monomer consists of three isostructural domains that coordinate and activate the three-fold symmetric substrate cyanuric acid for ring opening. We have now solved a series of X-ray structures of an engineered, thermostable cyanuric acid ring-opening enzyme at 1.51 ~ 2.25 Å resolution, including various complexes with the substrate, a tight-binding inhibitor, or an analog of the reaction intermediate. These structures reveal asymmetric interactions between the enzyme and bound ligands, a metal ion binding coupled to conformational changes and substrate binding important for enzyme stability, and distinct roles of the isostructural domains of the enzyme. The multiple conformations of the enzyme observed across a series of structures and corroborating biochemical data suggest importance of the structural dynamics in facilitating the substrate entry and the ring-opening reaction, catalyzed by a conserved Ser-Lys dyad.

Energetics for the Mechanism of Nickel-Containing Carbon Monoxide Dehydrogenase.

Nickel-containing carbon monoxide (CO) dehydrogenase is an enzyme that catalyzes the important reversible carbon dioxide reduction. Several high-resolution structures have been determined at various stages of the reduction, which can be used as good starting points for the present computational study. The cluster model is used in combination with a systematic application of the density functional theory as recently described. The results are in very good agreement with experimental evidence. There are a few important results. To explain why the X-ray structure for the reduced Cred1 state has an empty site on nickel, it is here suggested that the cluster has been over-reduced by X-rays and is therefore not the desired reduced state, which instead contains a bound CO on nickel. After an additional reduction, a hydride bound to nickel is suggested to play a role. In order to obtain energetics in agreement with experiments, it is concluded that one sulfide bridge in the Ni-Fe cluster should be protonated. The best test of the accuracy obtained is to compare the computed rate for reduction using -0.6 V with that for oxidation using -0.3 V, where good agreement was obtained. Obtaining a mechanism that is easily reversible is another demanding aspect of the modeling. Nickel oscillates between nickel(II) and nickel(I), while nickel(0) never comes in.

Binding site for coenzyme A revealed in the structure of pyruvate:ferredoxin oxidoreductase from Moorella thermoacetica.

Pyruvate:ferredoxin oxidoreductase (PFOR) is a microbial enzyme that uses thiamine pyrophosphate (TPP), three [4Fe-4S] clusters, and coenzyme A (CoA) in the reversible oxidation of pyruvate to generate acetyl-CoA and carbon dioxide. The two electrons that are generated as a result of pyruvate decarboxylation are used in the reduction of low potential ferredoxins, which provide reducing equivalents for central metabolism, including the Wood-Ljungdahl pathway. PFOR is a member of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily, which plays major roles in both microbial redox reactions and carbon dioxide fixation. Here, we present a set of crystallographic snapshots of the best-studied member of this superfamily, the PFOR from Moorella thermoacetica (MtPFOR). These snapshots include the native structure, those of lactyl-TPP and acetyl-TPP reaction intermediates, and the first of an OFOR with CoA bound. These structural data reveal the binding site of CoA as domain III, the function of which in OFORs was previously unknown, and establish sequence motifs for CoA binding in the OFOR superfamily. MtPFOR structures further show that domain III undergoes a conformational change upon CoA binding that seals off the active site and positions the thiolate of CoA directly adjacent to the TPP cofactor. These structural findings provide a molecular basis for the experimental observation that CoA binding accelerates catalysis by 105-fold.

Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073.

For the efficient production of target metabolites from carbohydrates, syngas, or H2-CO2 by genetically engineered Moorella thermoacetica, the control of acetate production (a main metabolite of M. thermoacetica) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica, this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 (pduL1) and Moth_1181 (pduL2), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 (T-ldh). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate.IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.

Two propanediol utilization-like proteins of Moorella thermoacetica with phosphotransacetylase activity.

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood-Ljungdahl (acetyl-CoA) pathway in which CO2 is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_0864 and moth_1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica. Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood-Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class(III)-PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).

One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography.

Thiamine pyrophosphate (TPP)-dependent oxalate oxidoreductase (OOR) metabolizes oxalate, generating two molecules of CO2 and two low-potential electrons, thus providing both the carbon and reducing equivalents for operation of the Wood-Ljungdahl pathway of acetogenesis. Here we present structures of OOR in which two different reaction intermediate bound states have been trapped: the covalent adducts between TPP and oxalate and between TPP and CO2. These structures, along with the previously determined structure of substrate-free OOR, allow us to visualize how active site rearrangements can drive catalysis. Our results suggest that OOR operates via a bait-and-switch mechanism, attracting substrate into the active site through the presence of positively charged and polar residues, and then altering the electrostatic environment through loop and side chain movements to drive catalysis. This simple but elegant mechanism explains how oxalate, a molecule that humans and most animals cannot break down, can be used for growth by acetogenic bacteria.

Cystathionine ß-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria.

Among numerous proteins containing pairs of regulatory cystathionine ß-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P(1),P(n)-diadenosine 5'-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ~10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.

Bacterial Cyanuric Acid Hydrolase for Water Treatment.

Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 µM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 µM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation.

The Structure of an Oxalate Oxidoreductase Provides Insight into Microbial 2-Oxoacid Metabolism.

Thiamine pyrophosphate (TPP), a derivative of vitamin B1, is a versatile and ubiquitous cofactor. When coupled with [4Fe-4S] clusters in microbial 2-oxoacid:ferredoxin oxidoreductases (OFORs), TPP is involved in catalyzing low-potential redox reactions that are important for the synthesis of key metabolites and the reduction of N2, H(+), and CO2. We have determined the high-resolution (2.27 Å) crystal structure of the TPP-dependent oxalate oxidoreductase (OOR), an enzyme that allows microbes to grow on oxalate, a widely occurring dicarboxylic acid that is found in soil and freshwater and is responsible for kidney stone disease in humans. OOR catalyzes the anaerobic oxidation of oxalate, harvesting the low-potential electrons for use in anaerobic reduction and fixation of CO2. We compare the OOR structure to that of the only other structurally characterized OFOR family member, pyruvate:ferredoxin oxidoreductase. This side-by-side structural analysis highlights the key similarities and differences that are relevant for the chemistry of this entire class of TPP-utilizing enzymes.

Evidence for a hexaheteromeric methylenetetrahydrofolate reductase in Moorella thermoacetica.

Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown.

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 ß 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 ß 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.

A reversible electron-bifurcating ferredoxin- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Moorella thermoacetica.

Moorella thermoacetica was long the only model organism used to study the biochemistry of acetogenesis from CO(2). Depending on the growth substrate, this Gram-positive bacterium can either form H(2) or consume it. Despite the importance of H(2) in its metabolism, a hydrogenase from the organism has not yet been characterized. We report here the purification and properties of an electron-bifurcating [FeFe]-hydrogenase from M. thermoacetica and show that the cytoplasmic enzyme efficiently catalyzes both H(2) formation and H(2) uptake. The purified heterotrimeric iron-sulfur flavoprotein (HydABC) catalyzed the coupled reduction of ferredoxin (Fd) and NAD(+) with H(2) at 55 °C at pH 7.5 at a specific rate of about 100 µmol min(-1) mg protein(-1) and the reverse reaction, the coupled reduction of protons to H(2) with reduced ferredoxin and NADH, at a specific rate of about 10 µmol min(-1) mg protein(-1) in the stoichiometry Fd(ox) + NAD(+) + 2H(2) Fd(red)(2-) + NADH + 3H(+). When ferredoxin from Clostridium pasteurianum, NAD(+), and the enzyme were incubated at pH 7.0 under 100% H(2) in the gas phase (E(0)' = -414 mV), more than 95% of the ferredoxin (E(0)' = -400 mV) was reduced, which indicated that ferredoxin reduction with H(2) is driven by the exergonic reduction of NAD(+) (E(0)' = -320 mV) with H(2). In the absence of NAD(+), ferredoxin was not reduced. We identified the genes encoding HydABC within the transcriptional unit hydCBAX and mapped the transcription start site.

Defining sequence space and reaction products within the cyanuric acid hydrolase (AtzD)/barbiturase protein family.

Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.

Visualizing molecular juggling within a B12-dependent methyltransferase complex.

Derivatives of vitamin B(12) are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO(2) fixation in acetogenic bacteria. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect and perform catalysis on the reactive B(12) cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes, inspiring speculation about the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete 220 kDa complex that contains all enzymes responsible for B(12)-dependent methyl transfer, namely the corrinoid iron-sulphur protein and its methyltransferase from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection and catalytic steps of B(12)-dependent methyl transfer. In addition, the structures capture B(12) at multiple locations between its 'resting' and catalytic positions, allowing visualization of the dramatic protein rearrangements that enable methyl transfer and identification of the trajectory for B(12) movement within the large enzyme scaffold. The structures are also presented alongside in crystallo spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.

Evidence that ferredoxin interfaces with an internal redox shuttle in Acetyl-CoA synthase during reductive activation and catalysis.

Acetyl-CoA synthase (ACS), a subunit of the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex of Moorella thermoacetica requires reductive activation in order to catalyze acetyl-CoA synthesis and related partial reactions, including the CO/[1-(14)C]-acetyl-CoA exchange reaction. We show that the M. thermoacetica ferredoxin(II) (Fd-II), which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. The level of activation depends on the oxidation states of both ACS and Fd-II, which strongly suggests that Fd-II acts as a reducing agent. By the use of controlled potential enzymology, the midpoint reduction potential for the catalytic one-electron redox-active species in the CO/acetyl-CoA exchange reaction is -511 mV, which is similar to the midpoint reduction potential that was earlier measured for other reactions involving ACS. Incubation of ACS with Fd-II and CO leads to the formation of the NiFeC species, which also supports the role of Fd-II as a reductant for ACS. In addition to being a reductant, Fd-II can accept electrons from acetylated ACS, as observed by the increased intensity of the EPR spectrum of reduced Fd-II, indicating that there is a stored electron within an "electron shuttle" in the acetyl-Ni(II) form of ACS. This "shuttle" is proposed to serve as a redox mediator during activation and at different steps of the ACS catalytic cycle.

Mutational analysis of residues in the regulatory CBS domains of Moorella thermoacetica pyrophosphatase corresponding to disease-related residues of human proteins.

mtCBS-PPase [CBS (cystathionine ß-synthase) domain-containing pyrophosphatase from Moorella thermoacetica] contains a pair of CBS domains that strongly bind adenine nucleotides, thereby regulating enzyme activity. Eight residues associated with the CBS domains of mtCBS-PPase were screened to explore possible associations with regulation of enzyme activity. The majority of the substitutions (V99A, R168A, Y169A, Y169F, Y188A and H189A) enhanced the catalytic activity of mtCBS-PPase, two substitutions (R170A and R187G) decreased activity, and one substitution (K100G) had no effect. AMP-binding affinity was markedly decreased in the V99A, R168A and Y169A mutant proteins, and elevated in the R187G and H189A mutant proteins. Remarkably, the R168A and Y169A substitutions changed the effect of AMP from inhibition to activation. The stoichiometry of AMP binding increased from one to two AMP molecules per CBS domain pair in the Y169F, R170A, R187G and Y188A variants. The ADP-binding affinity decreased in three and increased in four mutant proteins. These findings identify residues determining the strength and selectivity of nucleotide binding, as well as the direction (inhibition or activation) of the subsequent effect. The data suggest that mutations in human CBS domain-containing proteins can be translated into a bacterial context. Furthermore, our data support the hypothesis that the CBS domains act as an 'internal inhibitor' of mtCBS-PPase.