2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum.

Component D (HgdAB) of 2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum was purified to homogeneity. It is able to use component A from Acidaminococcus fermentans (HgdC) to initiate catalysis together with ATP, Mg2+ and a strong reducing agent such as Ti(III)citrate. Component D from C. symbiosum has a 6 x higher specific activity compared with that from A. fermentans and contains a second [4Fe-4S] cluster but the same amount of riboflavin 5'-phosphate (1.0 per heterodimeric enzyme, m = 100 kDa). M?ssbauer spectroscopy revealed symmetric cube-type structures of the two [4Fe-4S]2+ clusters. EPR spectroscopy showed the resistance of the clusters to reducing agents, but detected a sharp signal at g = 2. 004 probably due to a stabilized flavin semiquinone. Three genes from C. symbiosum coding for components D (hgdA and hgdB) and A (hgdC) were cloned and sequenced. Primer extension experiments indicated that the genes are transcribed in the order hgdCAB from an operon only half the size of that from A. fermentans. Sequence comparisons detected a close relationship to the dehydratase system from A. fermentans and HgdA from Fusobacterium nucleatum, as well as to putative proteins of unknown function from Archaeoglobus fulgidus. Lower, but significant, identities were found with putative enzymes from several methanogenic Archaea and Escherichia coli, as well as with the mechanistically related benzoyl-CoA reductases from the Proteobacteria Rhodopseudomonas palustris and Thauera aromatica.     

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.     

Substrate specificity of beta-collagenase from Clostridium histolyticum

The substrate specificity of beta-collagenase from Clostridium histolyticum has been investigated by measuring the rate of hydrolysis of more than 50 tri-, tetra-, penta-, and hexapeptides covering the P3 to P3' subsites of the substrate. The choice of peptides was patterned after sequences found in the alpha 1 and alpha 2 chains of type I collagen. Each peptide contained either a 2-furanacryloyl (FA) or cinnamoyl (CN) group in subsite P2 or the 4-nitrophenylalanine (Nph) residue in subsite P1. Hydrolysis of the P1-P1' bond produces an absorbance change in these chromophoric peptides that has been used to quantitate the rates of their hydrolysis under first order conditions ([S] much less than KM) from kcat/KM values have been obtained. The identity of the amino acids in all six subsites (P3-P3') markedly influences the hydrolysis rates. In general, the best substrates have Gly in subsites P3 and P1', Pro or Ala in subsite P2', and Hyp, Arg, or Ala in subsite P3'. This corresponds well with the frequency of occurrence of these residues in the Gly-X-Y triplets of collagen. In contrast, the most rapidly hydrolyzed substrates do not have residues from collagen-like sequences in subsites P2 and P1. For example, CN-Nph-Gly-Pro-Ala is the best known substrate for beta-collagenase with a kcat/KM value of 4.4 X 10(7) M-1 min-1, in spite of the fact that there is neither Pro nor Ala in P2 or Hyp nor Ala in P1. These results indicate that the previously established rules for the substrate specificity of the enzyme require modification.     

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].     

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.     

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).     

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.     

Substrate competition and specificity at the active site of amylopullulanase from Clostridium thermohydrosulfuricum

A highly thermostable pullulanase purified from Clostridium thermohydrosulfuricum strain 39E displayed dual activity with respect to glycosidic bond cleavage. The enzyme cleaved alpha-1,6 bonds in pullulan, while it showed alpha-1,4 activity against malto-oligosaccharides. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as the two competing substrates were used to distinguish the dual specificity of the enzyme from the single substrate specificity known for pullulanases and alpha-amylases.     

Substrate and stereochemical specificity of the organophosphorus acid anhydrolase from Alteromonas sp. JD6.5 toward p-nitrophenyl phosphotriesters

The enzyme OPAA hydrolyzes p-nitrophenyl phosphotriesters bearing substituents at the phosphorus center ranging in size from methyl to phenyl. The enzyme exhibits stereoselectivity toward the hydrolysis of chiral substrates with a preference for the Sp enantiomer.     

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.     

Substrate specificity of aromatic-L-amino acid decarboxylase

The benzo[b]thiophene and l-methylindole analogs of tryptophan have been tested for their ability to act as substrates for aromatic-L-amino acid decarboxylase $\text{in vitro}$. Neither of the analogs showed detectable decarboxylation under conditions that produced significant rates of decarboxylation of tryptophan. In addition, both analogs inhibited the decarboxylation of tryptophan and phenylalanine by the $\text{in vitro}$ system.     

The partial amino acid sequence of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum: implications for the evolution and structural basis of coenzyme specificity

The amino acid sequence is reported for CNBr and tryptic peptide fragments of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum. Together with the N-terminal sequence, these make up about 75% of the total sequence. The sequence shows extensive similarity with that of the NADP(+)-dependent glutamate dehydrogenase of Escherichia coli (52% identical residues out of the 332 compared) allowing confident placing of the peptide fragments within the overall sequence. This demonstrated sequence similarity with the E. coli enzyme, despite different coenzyme specificity, is much greater than the similarity (31% identities) between the GDH's of C. symbiosum and Peptostreptococcus asaccharolyticus, both NAD(+)-linked. The evolutionary implications are discussed. In the 'fingerprint' region of the nucleotide binding fold the sequence Gly X Gly X X Ala is found, rather than Gly X Gly X X Gly. The sequence found here has previously been associated with NADP+ specificity and its finding in a strictly NAD(+)-dependent enzyme requires closer examination of the function of this structural motif.     

Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase

The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho[(35)S]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-](n)(), resulted in 2-O-sulfation of iduronic acid. When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure [(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-](n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid. Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid. In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).     

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.     

Identification of domain-domain docking sites within Clostridium symbiosum pyruvate phosphate dikinase by amino acid replacement

Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosum apoPPDK, were replaced by site-directed mutagenesis. The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency. PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>10(4)) of the ATP pyrophosphorylation of His(455) at the N-terminal domain. In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover. Arg(219) and Glu(271) were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His(455) into position for catalysis. Substitution of central domain residues Glu(434)/Glu(437) and Thr(453), the respective docking partners of Arg(219) and Glu(271), resulted in mutants impaired in catalysis at the ATP active site. The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr(453)-Glu(271) binding interaction. With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH and Met(452) backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution.     

Substrate specificity of a dicarboxyl-CoA: dicarboxylic acid coenzyme A transferase from rat liver mitochondria.

Substrate specificity of a dicarboxyl-CoA: dicarboxylic acids coenzyme A transferase purified from rat liver mitochondria was assayed. In addition to the previously identified substrates succinyl-CoA, 3-hydroxy-3-methylglutaryl-CoA and malonyl-CoA (Francesconi et al.(1989) Biochim. Biophys. Acta, 999, 163-170) also methylmalonyl-CoA, glutaryl-CoA and adipyl-CoA acted as enzyme substrates, with the latter thioester showing the highest apparent affinity. All corresponding dicarboxylic acids, but not oxaloacetic, citric, alpha-ketoglutaric, malic, fumaric and glutamic acids, acted as coenzyme A acceptor substrates. None of the tested monocarboxylic acids, or the corresponding coenzyme A esters, were enzymatically transformed by the here described coenzyme A transferase.     

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 .