The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

Structure-Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme-Crystallographic Study of Glutamate Mutase with Cofactor Homologues.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012 -fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.

Photoproduct formation in coenzyme B12-dependent CarH via a singlet pathway.

The CarH photoreceptor exploits of the light-sensing ability of coenzyme B12 ( adenosylcobalamin = AdoCbl) to perform its catalytic function, which includes large-scale structural changes to regulate transcription. In daylight, transcription is activated in CarH via the photo-cleavage of the Co-C5' bond of coenzyme B12. Subsequently, the photoproduct, 4',5'-anhydroadenosine (anhAdo) is formed inducing dissociation of the CarH tetramer from DNA. Several experimental studies have proposed that hydridocoblamin (HCbl) may be formed in process with anhAdo. The photolytic cleavage of the Co-C5' bond of AdoCbl was previously investigated using photochemical techniques and the involvement of both singlet and triplet excited states were explored. Herein, QM/MM calculations were employed to probe (1) the photolytic processes which may involve singlet excited states, (2) the mechanism of anhAdo formation, and (3) whether HCbl is a viable intermediate in CarH. Time-dependent density functional theory (TD-DFT) calculations indicate that the mechanism of photodissociation of the Ado ligand involves the ligand field (LF) portion of the lowest singlet excited state (S1) potential energy surface (PES). This is followed by deactivation to a point on the S0 PES where the Co-C5' bond remains broken. This species corresponds to a singlet diradical intermediate. From this point, the PES for anhAdo formation was explored, using the Co-C5' and Co-C4' bond distances as active coordinates, and a local minimum representing anhAdo and HCbl formation was found. The transition state (TS) for the formation of the Co-H bond of HCbl was located and its identity was confirmed by a single imaginary frequency of i1592 cm-1. Comparisons to experimental studies and the potential role of rotation around the N-glycosidic bond of the Ado ligand were discussed.

Resolution and characterization of contributions of select protein and coupled solvent configurational fluctuations to radical rearrangement catalysis in coenzyme B12-dependent ethanolamine ammonia-lyase.

Coenzyme B12 (adenosylcobalamin) -dependent ethanolamine ammonia-lyase (EAL) is the signature enzyme in ethanolamine utilization metabolism associated with microbiome homeostasis and disease conditions in the human gut. The enzyme conducts a complex choreography of bond-making/bond-breaking steps that rearrange substrate to products through a radical mechanism, with themes common to other coenzyme B12-dependent and radical enzymes. The methods presented are targeted to test the hypothesis that particular, select protein and coupled solvent configurational fluctuations contribute to enzyme function. The general approach is to correlate enzyme function with an introduced perturbation that alters the properties (for example, degree of concertedness, or collectiveness) of protein and coupled solvent dynamics. Methods for sample preparation and low-temperature kinetic measurements by using temperature-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy are detailed. A framework for interpretation of results obtained in ensemble systems under conditions of statistical equilibrium within the reacting, globally unstable state is presented. The temperature-dependence of the first-order rate constants for decay of the cryotrapped paramagnetic substrate radical state in EAL, through the chemical step of radical rearrangement, displays a piecewise-continuous Arrhenius dependence from 203 to 295K, punctuated by a kinetic bifurcation over 219-220K. The results reveal the obligatory contribution of a class of select collective protein and coupled solvent fluctuations to the interconversion of two resolved, sequential configurational substates, on the decay time scale. The select class of collective fluctuations also contributes to the chemical step. The methods and analysis are generally applicable to other coenzyme B12-dependent and related radical enzymes.

Reactivating chaperones for coenzyme B12-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.

Coenzyme B12-dependent eliminases: Diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.

An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH.

Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.

Coenzyme B12 -dependent and independent photoregulation of carotenogenesis across Myxococcales.

Light-induced carotenogenesis in Myxococcus xanthus is controlled by the B12 -based CarH repressor and photoreceptor, and by a separate intricate pathway involving singlet oxygen, the B12 -independent CarH paralogue CarA and various other proteins, some eukaryotic-like. Whether other myxobacteria conserve these pathways and undergo photoregulated carotenogenesis is unknown. Here, comparative analyses across 27 Myxococcales genomes identified carotenogenic genes, albeit arranged differently, with carH often in their genomic vicinity, in all three Myxococcales suborders. However, CarA and its associated factors were found exclusively in suborder Cystobacterineae, with carA-carH invariably in tandem in a syntenic carotenogenic operon, except for Cystobacter/Melittangium, which lack CarA but retain all other factors. We experimentally show B12 -mediated photoregulated carotenogenesis in representative myxobacteria, and a remarkably plastic CarH operator design and DNA binding across Myxococcales. Unlike the two characterized CarH from other phyla, which are tetrameric, Cystobacter CarH (the first myxobacterial homologue amenable to analysis in vitro) is a dimer that combines direct CarH-like B12 -based photoregulation with CarA-like DNA binding and inhibition by an antirepressor. This study provides new molecular insights into B12 -dependent photoreceptors. It further establishes the B12 -dependent pathway for photoregulated carotenogenesis as broadly prevalent across myxobacteria and its evolution, exclusively in one suborder, into a parallel complex B12 -independent circuit.

Glycerol as a Substrate and Inactivator of Coenzyme B12 -Dependent Diol Dehydratase.

Diol dehydratase, dependent on coenzyme B12 (B12 -dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B12 -independent glycerol dehydratase (B12 -iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B12 -dDDH and that of resistance of B12 -iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B12 -dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B12 -iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.

The Biological Methane-Forming Reaction: Mechanism Confirmed Through Spectroscopic Characterization of a Key Intermediate.

Find your path: Methyl-coenzyme M reductase (MCR, turquoise) reversibly catalyzes the reduction of methyl-coenzyme M (methyl-S-CoM) with coenzyme B (CoB-SH) to form methane and the heterodisulfide. Recently, spectroscopic methods were used to detect trapped intermediates in a stopped-flow system, and CoM-S-NiII was identified after half a turnover of the MCR reaction (F430 =nickel porphinoid). This finding supports a methyl-radical catalytic mechanism.

The reaction mechanism of methyl-coenzyme M reductase: how an enzyme enforces strict binding order.

Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mM). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(Ni(I))·CH3SCoM) is highly favored (Kd = 79 µM). Only then can the chemical reaction occur (kobs = 20 s(-1) at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(Ni(II))·CoB7S(-)·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates.

Electron transport in acetate-grown Methanosarcina acetivorans.

BACKGROUND: Acetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H2 as an intermediate, although the great majority of acetotrophs are unable to metabolize H2. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H2-utilizing Methanosarcina acetivorans from H2-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens. RESULTS: A component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans. CONCLUSIONS: The results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H2-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.

Methyl-coenzyme M reductase from Methanothermobacter marburgensis.

Methyl-coenzyme M reductase catalyzes the reversible synthesis of methane from methyl-coenzyme M in methanogenic and ANME-1 and ANME-2 Archaea. The purification procedure for methyl-coenzyme M reductase from Methanothermobacter marburgensis is described. The procedure is an accumulation of almost 30 years of research on MCR starting with the first purification described by Ellefson and Wolfe (Ellefson, W.L., and Wolfe, R.S. (1981). Component C of the methylreductase system of Methanobacterium. J. Biol. Chem.256, 4259-4262). To provide a context for this procedure, some background information is provided, including a description of whole cell experiments that provided much of our knowledge of the behavior and properties of methyl-coenzyme M reductase.

2-oxoacid metabolism in methanogenic CoM and CoB biosynthesis.

Coenzyme M (CoM) and coenzyme B (CoB) are essential for methane production by the euryarchaea that employ this specialized anaerobic metabolism. Two pathways are known to produce CoM, 2-mercaptoethanesulfonate, and both converge on the 2-oxoacid sulfopyruvate. These cells have recruited the rich biochemistry of amino acid and 2-oxoacid metabolizing enzymes to produce a compound that resembles oxaloacetate, but with a more stable and acidic sulfonate group. 7-Mercaptoheptanoylthreonine phosphate, CoB, likewise owes its carbon backbone to a 2-oxoacid. Three enzymes recruited from leucine biosynthesis have evolved to catalyze the elongation of 2-oxoglutarate to 2-oxosuberate in CoB biosynthesis. This chapter describes the enzymology, synthesis, and analytical techniques used to study 2-oxoacid metabolism in these pathways. Protein structure and mechanistic information from enzymes provide insight into the evolution of new enzymatic activity, and the evolution of substrate specificity from promiscuous enzyme scaffolds.

Coupling of ferredoxin and heterodisulfide reduction via electron bifurcation in hydrogenotrophic methanogenic archaea.

In methanogenic archaea growing on H(2) and CO(2) the first step in methanogenesis is the ferredoxin-dependent endergonic reduction of CO(2) with H(2) to formylmethanofuran and the last step is the exergonic reduction of the heterodisulfide CoM-S-S-CoB with H(2) to coenzyme M (CoM-SH) and coenzyme B (CoB-SH). We recently proposed that in hydrogenotrophic methanogens the two reactions are energetically coupled via the cytoplasmic MvhADG/HdrABC complex. It is reported here that the purified complex from Methanothermobacter marburgensis catalyzes the CoM-S-S-CoB-dependent reduction of ferredoxin with H(2). Per mole CoM-S-S-CoB added, 1 mol of ferredoxin (Fd) was reduced, indicating an electron bifurcation coupling mechanism: 2H(2) + Fd(OX) + CoM-S-S-CoB-->Fd(red)(2-) + CoM-SH + CoB-SH + 2H(+). This stoichiometry of coupling is consistent with an ATP gain per mole methane from 4 H(2) and CO(2) of near 0.5 deduced from an H(2)-threshold concentration of 8 Pa and a growth yield of up to 3 g/mol methane.

Detection of organometallic and radical intermediates in the catalytic mechanism of methyl-coenzyme M reductase using the natural substrate methyl-coenzyme M and a coenzyme B substrate analogue.

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the terminal step in methanogenesis using coenzyme B (CoBSH) as the two-electron donor to reduce methyl-coenzyme M (methyl-SCoM) to form methane and the heterodisulfide, CoBS-SCoM. The active site of MCR contains an essential redox-active nickel tetrapyrrole cofactor, coenzyme F(430), which is active in the Ni(I) state (MCR(red1)). Several catalytic mechanisms have been proposed for methane synthesis that mainly differ in whether an organometallic methyl-Ni(III) or a methyl radical is the first catalytic intermediate. A mechanism was recently proposed in which methyl-Ni(III) undergoes homolysis to generate a methyl radical (Li, X., Telser, J., Kunz, R. C., Hoffman, B. M., Gerfen, G., and Ragsdale, S. W. (2010) Biochemistry 49, 6866-6876). Discrimination among these mechanisms requires identification of the proposed intermediates, none of which have been observed with native substrates. Apparently, intermediates form and decay too rapidly to accumulate to detectible amounts during the reaction between methyl-SCoM and CoBSH. Here, we describe the reaction of methyl-SCoM with a substrate analogue (CoB(6)SH) in which the seven-carbon heptanoyl moiety of CoBSH has been replaced with a hexanoyl group. When MCR(red1) is reacted with methyl-SCoM and CoB(6)SH, methanogenesis occurs 1000-fold more slowly than with CoBSH. By transient kinetic methods, we observe decay of the active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical intermediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformational changes in active Ni(I)-MCR(red1). Electron paramagnetic resonance (EPR) studies coupled with isotope labeling experiments demonstrate that the radical intermediate is not tyrosine-based. These observations provide support for a mechanism for MCR that involves methyl-Ni(III) and an organic radical as catalytic intermediates. Thus, the present study provides important mechanistic insights into the mechanism of this key enzyme that is central to biological methane formation.

Structural insight into methyl-coenzyme M reductase chemistry using coenzyme B analogues .

Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis: the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme and contains a highly reduced nickel-tetrapyrrole, coenzyme F(430). Methyl-SCoM must enter the active site prior to CoBSH, as species derived from methyl-SCoM are always observed bound to the F(430) nickel in the deepest part of the 30 A long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 A predominantly hydrophobic part of the channel close to F(430), with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 A. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues: pentanoyl, hexanoyl, octanoyl, and nonanoyl derivatives of CoBSH (CoB(5)SH, CoB(6)SH, CoB(8)SH, and CoB(9)SH, respectively). The data presented here reveal that the shorter CoB(5)SH mercaptoalkanoyl chain overlays with that of CoBSH but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB(6)SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB(6)SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB(8)SH and CoB(9)SH analogues can be accommodated in the active site through exclusion of this water. These analogues react with Ni(III)-methyl, a proposed MCR catalytic intermediate of methanogenesis. The CoB(8)SH thiolate is 2.6 A closer to the nickel than that of CoBSH, but the additional carbon of CoB(9)SH only decreases the nickel thiolate distance a further 0.3 A. Although the analogues do not induce any structural changes in the substrate channel, the thiolates appear to preferentially bind at two distinct positions in the channel, one being the previously observed CoBSH thiolate position and the other being at a hydrophobic annulus of residues that lines the channel proximal to the nickel.

Binding of coenzyme B induces a major conformational change in the active site of methyl-coenzyme M reductase.

Methyl-coenzyme M reductase (MCR) is the key enzyme in methane formation by methanogenic Archaea. It converts the thioether methyl-coenzyme M and the thiol coenzyme B into methane and the heterodisulfide of coenzyme M and coenzyme B. The catalytic mechanism of MCR and the role of its prosthetic group, the nickel hydrocorphin coenzyme F(430), is still disputed, and no intermediates have been observed so far by fast spectroscopic techniques when the enzyme was incubated with the natural substrates. In the presence of the competitive inhibitor coenzyme M instead of methyl-coenzyme M, addition of coenzyme B to the active Ni(I) state MCR(red1) induces two new species called MCR(red2a) and MCR(red2r) which have been characterized by pulse EPR spectroscopy. Here we show that the two MCR(red2) signals can also be induced by the S-methyl- and the S-trifluoromethyl analogs of coenzyme B. (19)F-ENDOR data for MCR(red2a) and MCR(red2r) induced by S-CF(3)-coenzyme B show that, upon binding of the coenzyme B analog, the end of the 7-thioheptanoyl chain of coenzyme B moves closer to the nickel center of F(430) by more than 2 A as compared to its position in both, the Ni(I) MCR(red1) form and the X-ray structure of the inactive Ni(II) MCR(ox1-silent) form. The finding that the protein is able to undergo a conformational change upon binding of the second substrate helps to explain the dramatic change in the coordination environment induced in the transition from MCR(red1) to MCR(red2) forms and opens the possibility that nickel coordination geometries other than square planar, tetragonal pyramidal, or elongated octahedral might occur in intermediates of the catalytic cycle.

Protein phosphorylation by semisynthesis: from paper to practice.

Deconvolution of specific phosphorylation events can be complicated by the reversibility of modification. Protein semisynthesis with phosphonate analogues offers an attractive approach to functional analysis of signaling pathways. In this technique, N- and C-terminal synthetic peptides containing nonhydrolyzable phosphonates at target residues can be ligated to recombinant proteins of interest. The resultant semisynthetic proteins contain site specific, stoichiometric phosphonate modifications and are completely resistant to phosphatases. Control of stoichiometry, specificity, and reversibility allows for complex signaling systems to be broken down into individual events and discretely examined. This chapter outlines the general methods and considerations for designing and carrying out phosphoprotein semisynthetic projects.

Methanogen homoaconitase catalyzes both hydrolyase reactions in coenzyme B biosynthesis.

Homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis. Despite the homology of this iron-sulfur protein to aconitase, previously studied homoaconitases catalyze only the hydration of cis-homoaconitate to form homoisocitrate rather than the complete isomerization of homocitrate to homoisocitrate. The MJ1003 and MJ1271 proteins from the methanogen Methanocaldococcus jannaschii formed the first homoaconitase shown to catalyze both the dehydration of (R)-homocitrate to form cis-homoaconitate, and its hydration is shown to produce homoisocitrate. This heterotetrameric enzyme also used the analogous longer chain substrates cis-(homo)(2)aconitate, cis-(homo)(3)aconitate, and cis-(homo)(4)aconitate, all with similar specificities. A combination of the homoaconitase with the M. jannaschii homoisocitrate dehydrogenase catalyzed all of the isomerization and oxidative decarboxylation reactions required to form 2-oxoadipate, 2-oxopimelate, and 2-oxosuberate, completing three iterations of the 2-oxoacid elongation pathway. Methanogenic archaeal homoaconitases and fungal homoaconitases evolved in parallel in the aconitase superfamily. The archaeal homoaconitases share a common ancestor with isopropylmalate isomerases, and both enzymes catalyzed the hydration of the minimal substrate maleate to form d-malate. The variation in substrate specificity among these enzymes correlated with the amino acid sequences of a flexible loop in the small subunits.