Substrate specificity of 2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum: toward a bio-based production of adipic acid

Expression of six genes from two glutamate fermenting clostridia converted Escherichia coli into a producer of glutaconate from 2-oxoglutarate of the general metabolism (Djurdjevic, I. et al. 2010, Appl. Environ. Microbiol.77, 320-322). The present work examines whether this pathway can also be used to reduce 2-oxoadipate to (R)-2-hydroxyadipic acid and dehydrate its CoA thioester to 2-hexenedioic acid, an unsaturated precursor of the biotechnologically valuable adipic acid (hexanedioic acid). 2-Hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum, the key enzyme of this pathway and a potential radical enzyme, catalyzes the reversible dehydration of (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA. Using a spectrophotometric assay and mass spectrometry, it was found that (R)-2-hydroxyadipoyl-CoA, oxalocrotonyl-CoA, muconyl-CoA, and butynedioyl-CoA, but not 3-methylglutaconyl-CoA, served as alternative substrates. Hydration of butynedioyl-CoA most likely led to 2-oxosuccinyl-CoA, which spontaneously hydrolyzed to oxaloacetate and CoASH. The dehydratase is not specific for the CoA-moiety because (R)-2-hydroxyglutaryl-thioesters of N-acetylcysteamine and pantetheine served as almost equal substrates. Whereas the related 2-hydroxyisocaproyl-CoA dehydratase generated the stable and inhibitory 2,4-pentadienoyl-CoA radical, the analogous allylic ketyl radical could not be detected with muconyl-CoA and 2-hydroxyglutaryl-CoA dehydratase. With the exception of (R)-2-hydroxyglutaryl-CoA, all mono-CoA-thioesters of dicarboxylates used in this study were synthesized with glutaconate CoA-transferase from Acidaminococcus fermentans. The now possible conversion of (R)-2-hydroxyadipate via (R)-2-hydroxyadipoyl-CoA and 2-hexenedioyl-CoA to 2-hexenedioate paves the road for a bio-based production of adipic acid.     

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

1. The (R)-2-hydroxyglutaryl-CoA dehydratase system from Acidaminococcus fermentans was separated by chromatography of cell-free extracts on Q-Sepharose into two components, an activator and the actual dehydratase. The latter enzyme was further purified to homogeneity by chromatography on blue-Sepharose. It is an iron-sulfur protein (Mr 210,000) consisting of two different polypeptides (alpha, Mr 55,000, and beta, Mr 42,000) in an alpha 2 beta 2 structure with probably two [4Fe-4S] centers. After activation this purified enzyme catalysed the dehydration of (R)-2-hydroxyglutarate only in the presence of acetyl-CoA and glutaconate CoA-transferase, demonstrating that the thiol ester and not the free acid is the substrate of the dehydration. The result led to a modification of the hydroxyglutarate pathway of glutamate fermentation. 2. The activation of the dehydratase by the flow-through from Q-Sepharose concentrated by ultrafiltration required NADH, MgCl2, ATP and strict anaerobic conditions. This fraction was designated as Ao. Later when the concentration was performed by chromatography on phenyl-Sepharose, an NADH-independent form of the activator, designated as A*, was obtained. This enzyme, which required only ATP for activation of the dehydratase, was purified further by affinity chromatography on ATP-agarose. It contains neither iron nor inorganic sulfur. A*, as well as the activated dehydratase, were irreversibly inactivated by exposure to air within less than 15 min. The activated dehydratase but not A* was also inactivated by 1 mM hydroxylamine or by 0.1 mM 2,4-dinitrophenol. 3. The (R)-2-hydroxyglutaryl-CoA dehydratase system is closely related the that of (R)-lactoyl-CoA dehydratase from Clostridium propionicum as described by R. D. Kuchta and R. H. Abeles [(1985) J. Biol. Chem. 260, 13,181-13,189].     

Adenosine triphosphate-induced electron transfer in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans

2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans catalyzes the chemical difficult elimination of water from (R)-2-hydroxyglutaryl-CoA to glutaconyl-CoA. The enzyme consists of two oxygen-sensitive protein components, the homodimeric activator (A) with one [4Fe-4S]1+/2+ cluster and the heterodimeric dehydratase (D) with one nonreducible [4Fe-4S]2+ cluster and reduced riboflavin 5'-monophosphate (FMNH2). For activation, ATP, Mg2+, and a reduced flavodoxin (16 kDa) purified from A. fermentans are required. The [4Fe-4S](1+/2+) cluster of component A is exposed to the solvent since it is accessible to iron chelators. Upon exchange of the bound ADP by ATP, the chelation rate is 8-fold enhanced, indicating a large conformational change. Oxidized component A exhibits ATPase activity of 6 s(-1), which is completely abolished upon reduction by one electron. UV-visible spectroscopy revealed a spontaneous one-electron transfer from flavodoxin hydroquinone (E(0)' = -430 mV) to oxidized component A, whereby the [4Fe-4S]2+ cluster of component A became reduced. Combined kinetic, EPR, and M?ssbauer spectrocopic investigations exhibited an ATP-dependent oxidation of component A by component D. Whereas the [4Fe-4S]2+ cluster of component D remained in the oxidized state, a new EPR signal became visible attributed to a d1-metal species, probably Mo(V). Metal analysis with neutron activation and atomic absorption spectroscopy gave 0.07-0.2 Mo per component D. In summary, the data suggest that in the presence of ATP one electron is transferred from flavodoxin hydroquinone via the [4Fe-4S]1+/2+ cluster of component A to Mo(VI) of component D, which is thereby reduced to Mo(V). The latter may supply the electron necessary for transient charge reversal in the unusual dehydration.     

The iron-sulfur clusters in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. Biochemical and spectroscopic investigations.

The reversible dehydration of (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA is catalysed by the combined action of two oxygen-sensitive enzymes from Acidaminococcus fermentans, the homodimeric component A (2 x 27 kDa) and the heterodimeric component D (45 and 50 kDa). Component A was purified to homogeneity (specific activity 25-30 s-1) using streptavidin-tag affinity chromatography. In the presence of 5 mM MgCl2 and 1 mM ADP or ATP, component A could be stabilized and stored for 4-5 days at 4 degrees C without loss of activity. The purification of component D from A. fermentans was also improved as indicated by the 1.5-fold higher specific activity (15 s-1). The content of 1.0 riboflavin 5'-phosphate (FMN) per heterodimer could be confirmed, whereas in contrast to an earlier report only trace amounts of riboflavin (< 0.1) could be detected. Each active component contains an oxygen sensitive diamagnetic [4Fe-4S]2+ cluster as revealed by UV-visible, EPR and M?ssbauer spectroscopy. Reduction of the [4Fe-4S]2+ cluster in component A with dithionite yields a paramagnetic [4Fe-4S]1+ cluster with the unusual electron spin ground state S = 3/2 as indicated by strong absorption type EPR signals at high g values, g = 4-6. Spin-Hamiltonian simulations of the EPR spectra and of magnetic M?ssbauer spectra were performed to determine the zero field splitting (ZFS) parameters of the cluster and the 57Fe hyperfine interaction parameters. The electronic properties of the [4Fe-4S]2+, 1+ clusters of component A are similar to those of the nitrogenase iron protein in which a [4Fe-4S]2+ cluster bridges the two subunits of the homodimeric protein. Under air component A looses its activity within seconds due to irreversible degradation of its [4Fe-4S]2+ cluster to a [2Fe-2S]2+ cluster. The [4Fe-4S]2+ cluster of component D could not be reduced to a [4Fe-4S]1+ cluster, even with excess of Ti(III)citrate or dithionite. Exposure to oxic conditions slowly converts the diamagnetic [4Fe-4S]2+ cluster of component D to a paramagnetic [3Fe-4S]+ cluster concomitant with loss of activity (30% within 24 h at 4 degrees C).     

Crystal structure of the Acidaminococcus fermentans 2-hydroxyglutaryl-CoA dehydratase component A

Acidaminococcus fermentans degrades glutamate via the hydroxyglutarate pathway, which involves the syn-elimination of water from (R)-2-hydroxyglutaryl-CoA in a key reaction of the pathway. This anaerobic process is catalyzed by 2-hydroxyglutaryl-CoA dehydratase, an enzyme with two components (A and D) that reversibly associate during reaction cycles. Component A (CompA), a homodimeric protein of 2x27 kDa, contains a single, bridging [4Fe-4S] cluster and uses the hydrolysis of ATP to deliver an electron to the dehydratase component (CompD), where the electron is used catalytically. The structure of the extremely oxygen-sensitive CompA protein was solved by X-ray crystallography to 3 A resolution. The protein was found to be a member of the actin fold family, revealing a similar architecture and nucleotide-binding site. The key differences between CompA and other members of the actin fold family are: (i) the presence of a cluster binding segment, the "cluster helix"; (ii) the [4Fe-4S] cluster; and (iii) the location of the homodimer interface, which involves the bridging cluster. Possible reaction mechanisms are discussed in light of the close structural similarity to members of the actin-fold family and the functional similarity to the nitrogenase Fe- protein.     

2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile

The hadBC and hadI genes from Clostridium difficile were functionally expressed in Escherichia coli and shown to encode the novel 2-hydroxyisocaproyl-CoA dehydratase HadBC and its activator HadI. The activated enzyme catalyses the dehydration of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA in the pathway of leucine fermentation. The extremely oxygen-sensitive homodimeric activator as well as the heterodimeric dehydratase, contain iron and inorganic sulfur; besides varying amounts of zinc, other metal ions, particularly molybdenum, were not detected in the dehydratase. The reduced activator transfers one electron to the dehydratase concomitant with hydrolysis of ATP, a process similar to that observed with the unrelated nitrogenase. The thus activated dehydratase was separated from the activator and ATP; it catalyzed about 10(4) dehydration turnovers until the enzyme became inactive. Adding activator, ATP, MgCl(2), dithionite and dithioerythritol reactivated the enzyme. This is the first demonstration with a 2-hydroxyacyl-CoA dehydratase that the catalytic electron is recycled after each turnover. In agreement with this observation, only substoichiometric amounts of activator (dehydratase/activator = 10 mol/mol) were required to generate full activity.     

Crystallization of an NAD+-dependent glutamate dehydrogenase from Clostridium symbiosum

Crystals of a bacterial NAD+-dependent glutamate dehydrogenase (GDHase) have been grown over a wide range of pH values by using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of this enzyme together with high pressure liquid chromatography/gel filtration, shows that this GDHase is hexameric like the GDHases of vertebrates. X-ray photographs of the crystals show that they diffract to at least 2.0 A, and an analysis of the diffraction pattern demonstrates that the hexamer is arranged in at least pseudo 32 symmetry.     

Characterization of the surface layer glycoprotein of Clostridium symbiosum HB25.

The cell surface of Clostridium symbiosum HB25 is covered by a squarely arranged surface layer (S-layer) glycoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the sodium dodecyl sulfate-soluble whole-cell extract showed the presence of several high-molecular-weight protein bands in a narrow range (approximate Mr, 140,000) which, upon periodic acid-Schiff staining, gave a positive reaction. After proteolytic degradation of the purified S-layer glycoprotein, a single glycopeptide fraction was obtained by gel permeation chromatography. Hydrolysis, treatment with aqueous hydrofluoric acid, and 1H and 13C nuclear magnetic resonance studies showed that the glycoprotein glycan is a high-molecular-weight polymer (approximate Mr, 15,000) of tetrasaccharide repeating units with the component sugars N-acetylgalactosamine (GalNAc), N-acetylmannosamine (ManNAc), and N-acetylbacillosamine (BacNAc; 2-N-acetyl-4-amino-2,4,6-trideoxy glucose) linked by monophosphate diesters. The following structure is proposed: [----6)-alpha-D-ManpNAc-(1----4)-beta-D-GalpNAc-(1----3)-alpha-D-+ ++BacpNAc- (1----4)-alpha-D-GalpNAc-(1----PO3)----]n. The nuclear magnetic resonance data provided evidence for a charge interaction between the free amino group of BacNAc and the phosphate group of adjacent glycan chains. Since polycationic ferritin did not label the cell surface of intact cells, an electrostatic interaction can also be expected in vivo, leading to a charge-neutral outer surface, which is characteristic of all other S layers from members of the family Bacillaceae studied so far.     

A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum.

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.     

Assay of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum

It has been proposed that Clostridium aminobutyricum contains an enzyme catalyzing an unusual reaction: the dehydration of 4-hydroxybutyryl-CoA to vinylacetyl-CoA. 4-Hydroxy-[3-3H]butyric acid has been prepared which allows the activity of this enzyme to be assayed in the presence of acetyl-CoA under anaerobic conditions by the release of tritiated water. Initial characterization of the enzyme from C. aminobutyricum has shown it to be largely membrane or particle bound in the crude lysates. It can be solubilized in detergent. It is inactivated by oxygen, but stable under anaerobic conditions. Only 49 +/- 2% of the label is removed after enzyme-catalyzed equilibration with water. This stereospecific release is consistent with the formation of vinylacetyl-CoA and excludes a vitamin B12 coenzyme-dependent rearrangement to 3-hydroxybutyryl-CoA followed by dehydration to crotonyl-CoA.     

Crystalline green 5-hydroxyvaleryl-CoA dehydratase from Clostridium aminovalericum

A green enzyme from Clostridium aminovalericum with valeryl-CoA dehydrogenase activity was purified to homogeneity (169 +/- 3 kDa) and crystallized. By SDS/PAGE, one type of subunit (42 kDa) was detected indicating a homotetrameric structure. The unusual ultraviolet/visible spectrum of the green enzyme (maxima at 394 nm, 438 nm and 715 nm) was converted to a normal flavoprotein spectrum either by reduction with dithionite and reoxidation under air, or by removal of the prosthetic group at pH 2 and reconstitution with FAD (not FMN). Besides FAD (4 mol/169 kDa), the enzyme contained 4 mol of a CoA ester which was similar but not identical to 5-hydroxy-2-pentenoyl-CoA. The reconstituted holoenzyme as well as the native green enzyme, but not the apoenzyme, catalysed the reversible dehydration of 5-hydroxyvaleryl-CoA to 4-pentenoyl-CoA in the absence of an external electron acceptor. In its presence (preferentially ferricenium ion), the green or yellow enzyme catalysed the formation of (E)-5-hydroxy-2-pentenoyl-CoA and 2,4-pentadienoyl-CoA either from 4-pentenoyl-CoA or from 5-hydroxyvaleryl-CoA. The reversible hydration of 2,4-pentadienoyl-CoA to (E)-5-hydroxy-2-pentenoyl-CoA was mediated by both enzymes as well as by the apoenzyme in the absence of FAD. Hydration of 4-pentenoate in 2H2O yielded optically active 5-hydroxy[2,4-2H2]valerate by the combined action of 5-hydroxyvalerate CoA-transferase, the green dehydratase and catalytical amounts of acetyl-CoA. The data show that the reversible hydration of the isolated double bond of 4-pentenoyl-CoA to 5-hydroxyvaleryl-CoA. which apparently violates the Markovnikov rule, is preceded by oxidation to 2,4-pentadienoyl-CoA. The latter compound, a vinyl analogue of 2-enoyl-CoA, is then easily hydrated to (E)-5-hydroxy-2-pentenoyl-CoA and finally reduced to 5-hydroxyvaleryl-CoA.     

Effect of additives on the crystallization of glutamate dehydrogenase from Clostridium symbiosum. Evidence for a ligand-induced conformational change.

A new crystal form of the hexameric NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum has been grown using the hanging drop method of vapour diffusion. The crystals are obtained either by using high concentrations of the amino acid substrate of the enzyme, glutamate, as the precipitant or by co-crystallization from ammonium sulphate in the presence of either p-chloromercuribenzene sulphonate or potassium tetracyanoplatinate. The crystals diffract well and X-ray photographs have established that they are in the space group R32. Considerations of the values of Vm indicate that the asymmetric unit of the R32 crystals contains a single subunit. Packing considerations based on the structure of the native enzyme determined from a different crystal form suggest that the molecule must undergo a significant conformational change in order to be accommodated in the new cell. Such a conformational rearrangement may represent an important step in the catalytic cycle.     

Clostridium symbiosum as a cause of bloodstream infection in an immunocompetent patient

Bloodstream infection caused by Clostridium symbiosum was previously reported in only one case, from a highly immunocompromised patient with metastic colon cancer. We describe the second case of clinical bacteraemic sepsis caused by C. symbiosum from a previously healthy man, which underlines the pathogenicity of this species.     

Lactate reduction in Clostridium propionicum. Purification and properties of lactyl-CoA dehydratase

Clostridium propionicum converts lactate to propionate (Cardon, B.P., and Barker, H.A. (1947) Arch. Biochem. Biophys. 12, 165-171). We have obtained a soluble system that carries out this conversion as well as the hydration of acrylate to lactate and the reduction of acrylate to propionate. 3-Pentynyl-CoA inhibits reduction of acrylate and lactate to propionate, but not hydration of acrylate to lactate by cell extracts. The conversion probably involves CoA esters. When [beta-2H3] lactate is used as a substrate, the rate of propionate formation is reduced 1.8-fold, and the methyl group of the resulting propionate has lost 1.4 deuterium atoms. These results are consistent with the intermediate formation of acrylate (acrylyl-CoA) in the conversion of D-lactate to propionate. Two proteins, which we designate E I and E II, were purified to greater than 90% homogeneity. Together, they catalyze the hydration of acrylyl-CoA to lactyl-CoA. E I has an apparent molecular mass of 27,000 daltons and is rapidly and irreversibly inactivated by O2. E II consists of two subunits of molecular mass 41,000 and 48,000 daltons and contains equal amounts of riboflavin and flavin mononucleotide. Hydration of acrylyl-CoA to lactyl-CoA requires Mg2+ and catalytic quantities of ATP. GTP can replace ATP, but ADP and adenylyl imidodiphosphate cannot. We were unable to detect any stable intermediate during acrylyl-CoA hydration. Finally, we proposed a mechanism for this reaction.     

Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces

X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer. Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme. The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis. The latter technique gives an s_20,w value for D165H of (6.07 ± 0.01)S which compares with (11.08 ± 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments. Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification. The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme. Accepted: 11 November 1996     

Morphology of Bacteriophage-Like Particles from Fusobacterium symbiosum

The morphology of bacteriophage-like particles from the strict anaerobe Fusobacterium symbiosum is described. Attempts to demonstrate plaque formation on the host strain of F. symbiosum and other related species were unsuccessful.     

Investigation of the role of the domain linkers in separate site catalysis by Clostridium symbiosum pyruvate phosphate dikinase.

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible reaction: ATP + P(i) + pyruvate <--> AMP + PP(i) + PEP using Mg2+ and NH4+ ions as cofactors. The reaction takes place in three steps, each mediated by a carrier histidine residue located on the surface of the central domain of this three-domain enzyme: (1) E-His + ATP <--> E-His-PP.AMP, (2) E-His-PP.AMP + P(i) <--> E-His-P + AMP + PP(i), (3) E-His-P + pyruvate <--> E-His + PEP. The first two partial reactions are catalyzed at an active site located on the N-terminal domain, and the third partial reaction is catalyzed at an active site located on the C-terminal domain. For catalytic turnover, the central domain travels from one terminal domain to the other. The goal of this work is to determine whether the two connecting linkers direct the movement of the central domain between active sites during catalytic turnover. The X-ray crystal structure of the enzyme suggests interaction between the two linkers that may result in their coordinated movement. Mutations were made at the linkers for the purpose of disrupting the linker-linker interaction and, hence, synchronized linker movement. Five linker mutants were analyzed. Two of these contain 4-Ala insertions within the solvated region of the linker, and three have 3-residue deletions in this region. The efficiencies of the mutants for catalysis of the complete reaction as well as the E-His + ATP <--> E-His-PP.AMP partial reaction at the N-terminal domain and the E-His + PEP <--> E-His-P + pyruvate reaction at the C-terminal domain were measured to assess linker function. Three linker mutants are highly active catalysts at both active sites, and the fourth is highly active at one site but not the other. These results are interpreted as evidence against coordinated linker movement, and suggest instead that the linkers move independently as the central domain travels between active sites. It is hypothesized that while the linkers play a passive role in central domain-terminal domain docking, their structural design minimizes the conformational space searched in the diffusion process.     

Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2) E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate --> E.PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/P(i) active site, to identify the residues at the ATP/P(i) active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + P(i) --> E-P + AMP + PP(i) partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, P(i), and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors. The P(i) is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.     

Ultrastructure of the cell envelope and amino acid composition of the murein of Clostridium symbiosum

Abstract The cell envelope of the Gram-negative staining Clostridium symbiosum is 18 nm thick. It appears triple-layered and consists of an inner electrondense layer of about 5 nm, a lighter zone of 4 nm and an outer electron-dense layer of 9 nm. The inner layer corresponds to the murein sacculus, since the isolated peptidoglycan sacculi showed a thickness of 3–5 nm. Analysis showed that it belongs to the A₂pm-direct murein type. The outer layer could be removed by sodium dodecylsulfate. It contained mainly protein, small amounts of sugars and essentially no lipid, indicative of an S-layer rather than a typical Gram-negative type of outer membrane. Furthermore, l -alanine aminopeptidase activity characteristic of Gram-negative aerobic bacteria was absent in this organism and in other anaerobic Gram-negative bacteria tested. This demonstrates that such activity is an unreliable tool for the classification of anaerobic eubacteria. In spite of the thin murein layer, which is the likely reason for the Gram-negative reaction, the anaerobic growth, peritrichous flagellation and endospore formation indicate that this organism belongs to the genus Clostridium .