Clostridium Symbiosum Research Articles

Clostridium symbiosumIsolated from Blood

Clostridium symbiosum was isolated from the blood of a 61-year-old immunocompromised woman who had diagnosed ovarian cancer with multiple metastases and who had developed persistent tachycardia. A blood culture was drawn from the peripherally inserted central catheter, and non-spore-forming gram-negative rods were detected in an anaerobic vial. The organism showed tiny and pinpoint colonies and was unidentified by Vitek II (bioMerieux, France). The 16S rRNA gene sequence showed a 99.4% identity with C. symbiosum. To our knowledge, this represents the first report of C. symbiosum isolation in Korea. (Korean J Clin Microbiol 2010;13: 90-92)

An Asymmetric Model for Na+-translocating Glutaconyl-CoA Decarboxylases

Glutaconyl-CoA decarboxylase (Gcd) couples the biotin-dependent decarboxylation of glutaconyl-CoA with the generation of an electrochemical Na(+) gradient. Sequencing of the genes encoding all subunits of the Clostridium symbiosum decarboxylase membrane complex revealed that it comprises two distinct biotin carrier subunits, GcdC(1) and GcdC(2), which differ in the length of a central alanine- and proline-rich linker domain. Co-crystallization of the decarboxylase subunit GcdA with the substrate glutaconyl-CoA, the product crotonyl-CoA, and the substrate analogue glutaryl-CoA, respectively, resulted in a high resolution model for substrate binding and catalysis revealing remarkable structural changes upon substrate binding. Unlike the GcdA structure from Acidaminococcus fermentans, these data suggest that in intact Gcd complexes, GcdA is associated as a tetramer crisscrossed by a network of solvent-filled tunnels.

Modular coenzyme specificity: A domain‐swopped chimera of glutamate dehydrogenase

Domain-swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD(+)-specific) and Escherichia coli (EcGDH) (NADP(+)-specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate-binding domain (Domain I) of CsGDH and the coenzyme-binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain II; the chimera utilizes NAD(+) at 0.8% of the rate observed with NADP(+), similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L-glutamate, characteristic of CsGDH, has been retained with Domain I. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher pH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V(max)) in both directions than either of the parent enzymes from which it is constructed.

本邦で認知度が低い<I>Clostndium</I>の感染症材料からの分離状況と薬剤感受性

Cigar-shaped clostridia such as Clostridium clostridioforme and Clostridium symbiosum have been found in serious infections such as bacteremia. In Japan, however, these strains are unrecognized as clinically significant because they are overlooked as unidentified gram-negative rods. We isolated 60 strains of cigar-shaped Clostridium spp. from 48 clinical specimens treated at our laboratory from November 2004 to September 2006. Of these, 19 (39.6%) were primary infections and 29 (60.4%) postsurgical. Primary infections included infected decubitus ulcer (7), peritonitis, intra abdominal abscess, sepsis (2 cases in each group), pyometra, Bartholin's gland abscess, and Fournier's gangrene (1 case in each group). Secondary infections included 28 cases of surgical site infection and 1 case of pyothorax. CRP in 30 cases (62.5%) exeeded 10 mg/dL. In 26 (54.2%), WBC exceeded 12,000/microL. The 60 isolated strains were as follows by 16S rRNA sequencing: Clostridium hathewayi (26.7%), C. clostridioforme (16.7%), Clostridium bolteae (18.3%), Clostridium citroniae (10%), Clostridium aldenense (8.3%), and Clostridium symbiosum (20%). In antimicrobial susceptibility, strains of C. hathe-wayi showed relatively higher MIC for cefotaxime (MIC50; 64 microg/mL, MIC90; 128 microg/mL). Three strains of C. bolteae were beta-lactamase-producing and their MICs for ampicillin exceeded 128 microg/mL.

Heat Capacity, Configurational Entropy, and the Role of Ionic Interactions in Protein Thermostability

ABSTRACTMolecular dynamics simulation of solvated proteins at different temperatures can provide valuable descriptions of their global dynamics and their local fluctuations that can deepen our understanding of the molecular basis of protein thermostability. In this work a comparison of the dynamical behavior of homologous glutamate dehydrogenase domains from the hyperthermophilic bacterium Thermotoga maritima and the mesophilic bacterium Clostridium symbiosum as well as a molecular dynamics simulation study of a short polypeptide designed to fold into a trimeric coiled coil are reviewed with particular emphasis on the contribution of ion pairs and ionic networks to the conformational stability of the proteins. By considering the solvated proteins in their dynamic context thermodynamic properties such as electrostatic free energy differences, heat capacity and configurational entropy were calculated and suggested a new way of understanding protein thermostability on the basis of energetic fluctuations and accessible states. It appeared that a larger number of accessible degrees of freedom seems to accommodate the disorder caused by increased thermal motion.

Swiveling Domain Mechanism in Pyruvate Phosphate Dikinase,

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.

Cooperativity of GDH from Clostridium symbiosum at neutral pH

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.

The contribution of tryptophan residues to conformational changes in clostridial glutamate dehydrogenase − W64 and W449 as mediators of the cooperative response to glutamate

The hexameric glutamate dehydrogenase of Clostridium symbiosum has previously been shown to undergo a pH-dependent inactivating conformational change that perturbs the environment of one or more Trp residues and is reversed by glutamate in a highly cooperative fashion with a Hill coefficient of almost 6. Five single mutants have now been made in which each of the Trp residues in turn has been replaced by Phe. All five were successfully over-produced as soluble proteins and purified. Far-UV CD showed that none of the mutations significantly affected secondary structure. All five proteins were active, ranging from 13 U.mg(-1) (W64F) to 20.8 U.mg(-1) (W393F), compared to 20 U.mg(-1) for wild-type, and the kinetic parameters at pH 7 were little changed, except for a five- to six-fold increase in Km for glutamate in W243F. Thermostability was also relatively little changed, although W310F and W393F were somewhat more stable and W64F less stable than the unmutated enzyme. All still showed the characteristic reversible, time-dependent high-pH inactivation. Near-UV CD spectra, reflecting the environment of aromatic residues, were recorded at both pH 7 and 8.8, and four of the mutants showed essentially the same perturbation in the 280 nm region as the wild-type enzyme. W64F, however, showed essentially no change. W64 is thus clearly a passive reporter of the pH-dependent conformational change, and not actually required for the transition to occur. The CD comparisons also suggest that the aromatic CD spectrum is contributed almost entirely by W64 and W449. Consistent with the pH-dependent change, all five mutant proteins also showed a positively cooperative response to glutamate at pH 9, reversing the inactivation. However, the Hill coefficient decreased from > 5 for wild-type to approximately 3 for the active site cleft mutation W243F and to approximately 2 for the interfacial mutants W64F and W449F in which the trimer-trimer interaction may be directly interrupted. W64 of each subunit is in contact with W449 in its dimer partner at the trimer-trimer interface. It seems that, although neither of these two residues is required for the pH-dependent change, together, they are essential in mediating the total cooperativity of the hexameric enzyme's response to glutamate and are presumably directly involved in transmitting conformational information between the two trimers.

Enhancing long-term thermal stability in mesophilic glutamate dehydrogenase from Clostridium symbiosum by eliminating cysteine residues

Glutamate dehydrogenase from Clostridium symbiosum has two cysteine residues, C144 and C320. The single mutant C320S and a double mutant with both cysteines replaced by serine have been compared with one another in terms of long-term stability and other properties. Specific activities and kinetic parameters were relatively little affected, but stability was improved—e.g. at 25 °C sterile, sealed samples of wild-type enzyme, C320S and the double mutant at 0.1 mg/ml in 0.1 M phosphate buffer, pH 7 lost 50%, 42% and 32% of activity over 60 days. For the first two proteins this loss was partly reversible with dithiothreitol. When wild-type enzyme was deliberately contaminated with 1 μM Cu 2+ it became less stable and formed aggregates, whereas the double mutant was not affected. The double mutation thus removes a source of instability through –SH oxidation that would be accentuated by any heavy metal contamination of solutions.

Clostridum 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.

Crystal Structures of Pyruvate Phosphate Dikinase from Maize Revealed an Alternative Conformation in the Swiveling-Domain Motion,

Pyruvate phosphate dikinase (PPDK) reversibly catalyzes the conversion of ATP, phosphate, and pyruvate into AMP, pyrophosphate, and phosphoenolpyruvate (PEP), respectively. Since the nucleotide binding site (in the N-terminal domain) and the pyruvate/PEP binding site (in the C-terminal domain) are separated by approximately 45 A, it has been proposed that an intermediary domain, called the central domain, swivels between these remote domains to transfer the phosphate. However, no direct structural evidence for the swiveling central domain has been found. In this study, the crystal structures of maize PPDK with and without PEP have been determined at 2.3 A resolution. These structures revealed that the central domain is located near the pyruvate/PEP binding C-terminal domain, in contrast to the PPDK from Clostridium symbiosum, wherein the central domain is located near the nucleotide-binding N-terminal domain. Structural comparisons between the maize and C. symbiosum PPDKs demonstrated that the swiveling motion of the central domain consists of a rotation of at least 92 degrees and a translation of 0.5 A. By comparing the maize PPDK structures with and without PEP, we have elucidated the mode of binding of PEP to the C-terminal domain and the induced conformational changes in the central domain.

Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology-based modelling.

Through comparison with the high-resolution structure of Clostridium symbiosum glutamate dehydrogenase, the different substrate specificities of the homologous enzymes phenylalanine dehydrogenase and leucine dehydrogenase were attributed to two residues, glycine 124 and leucine 307, in Bacillus sphaericus phenylalanine dehydrogenase, which are replaced with alanine and valine in leucine dehydrogenases. As predicted, making these substitutions in phenylalanine dehydrogenase decreased the specific activity towards aromatic substrates and enhanced the activity towards some aliphatic amino acids in standard assays with fixed concentrations of both substrates. This study did not, however, distinguish effects on affinity from those on maximum catalytic rate. A fuller kinetic characterization of the single- and double-mutant enzymes now reveals that the extent of the shift in specificity was underestimated in the earlier study. The maximum catalytic rates for aromatic substrates are reduced for all the mutants, but, in addition, the apparent Km values are higher for the single-mutant G124A and double-mutant G124A/L307V compared with the wild-type enzyme. Conversely, specificity constants (kcat/Km) for the nonpolar aliphatic amino acids and the corresponding 2-oxoacids for the mutants are all markedly higher than for the wild type, with up to a 40-fold increase for l-norvaline and a 100-fold increase for its 2-oxoacid in the double mutant. In some cases a favourable change in Km was found to outweigh a smaller negative change in kcat. These results emphasize the risk of misjudging the outcome of protein engineering experiments through too superficial an analysis. Overall, however, the success of the predictions from molecular modelling indicates the usefulness of this strategy for engineering new specificities, even in advance of more detailed 3D structural information.

A Thermally Sensitive Loop in Clostridial Glutamate Dehydrogenase Detected by Limited Proteolysis

The structural flexibility and thermostability of glutamate dehydrogenase (GDH) from Clostridium symbiosum were examined by limited proteolysis using three proteinases with different specificities, trypsin, chymotrypsin, and endoproteinase Glu-C. Clostridial GDH resisted proteolysis by any of these enzymes at 25 degrees C. Above 30 degrees C, however, GDH became cleavable by chymotrypsin, apparently at a single site. SDS-PAGE indicated the formation of one large fragment with a molecular mass of approximately 44 kDa and one small one of <10 kDa. Proteolysis was accompanied by the loss of enzyme activity, which outran peptide cleavage, suggesting a cooperative conformational change. Proteolysis was prevented by either of the substrates 2-oxoglutarate or l-glutamate but not by the coenzymes NAD(+) or NADH. Circular dichroism spectroscopy indicated that the protective effects of these ligands resulted from fixation of flexible regions of the native structure of the enzyme. Size-exclusion chromatography and SDS-PAGE studies of chymotrypsin-treated GDH showed that the enzyme retained its hexameric structure and all of its proteolytic fragments. However, circular dichroism spectroscopy and analytical ultracentrifugation showed global conformational changes affecting the overall compactness of the protein structure. Chymotrypsin-catalyzed cleavage also diminished the thermostability of GDH and the cooperativity of the transition between its native and denatured states. N-terminal amino acid sequencing and mass spectrometry showed that heat-induced sensitivity to chymotrypsin emerged in the loop formed by residues 390-393 that lies between helices alpha(15) and alpha(16) in the folded structure of the enzyme.

Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates.

Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively. The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine. This higher specificity toward L-phenylalanine was due to K(m) values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine. The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K(m) for this substrate. Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes. As with phenylalanine, these increases are mainly attributable to large decreases in K(m) values.

The 3.0 Å Resolution Crystal Structure of Glycosomal Pyruvate Phosphate Dikinase from Trypanosoma brucei

The crystal structure of the glycosomal enzyme pyruvate phosphate dikinase from the African protozoan parasite Trypanosoma brucei has been solved to 3.0Å resolution by molecular replacement. The search model was the 2.3Å resolution structure of the Clostridium symbiosum enzyme. Due to different relative orientations of the domains and sub-domains in the two structures, molecular replacement could be achieved only by positioning these elements (four bodies altogether) sequentially in the asymmetric unit of the P21212 crystal, which contains one pyruvate phosphate dikinase (PPDK) subunit. The refined model, comprising 898 residues and 188 solvent molecules per subunit, has a crystallographic residual index Rf=0.245 (cross-validation residual index Rfree=0.291) and displays satisfactory stereochemistry. Eight regions, comprising a total of 69 amino acid residues at the surface of the molecule, are disordered in this crystal form. The PPDK subunits are arranged around the crystallographic 2-fold axis as a dimer, analogous to that observed in the C.symbiosum enzyme. Comparison of the two structures was carried out by superposition of the models. Although the fold of each domain or sub-domain is similar, the relative orientations of these constitutive elements are different in the two structures. The trypanosome enzyme is more “bent” than the bacterial enzyme, with bending increasing from the center of the molecule (close to the molecular 2-fold axis) towards the periphery where the N-terminal domain is located. As a consequence of this increased bending and of the differences in relative positions of sub-domains, the nucleotide-binding cleft in the amino-terminal domain is wider in T.brucei PPDK: the N-terminal fragment of the amino-terminal domain is distant from the catalytic, phospho-transfer competent histidine 482 (ca 10Å away). Our observations suggest that the requirements of domain motion during enzyme catalysis might include widening of the nucleotide-binding cleft to allow access and departure of the AMP or ATP ligand.

Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD+–agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40mM glutamate and 1mM NAD+ at pH8.8, or K89L/A163G/S380A with 70mM norleucine and 1mM NAD+ at pH8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC50 = 70mM, h = 2.4), and glutarate gave approx. 30-fold activation (EC50 = 35mM, h = 2.3). For the triple mutant, corresponding values were 80mM and 2.2 for succinate, and 75mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The ‘foreign’ subunit evidently impedes inter-subunit communication to some extent.

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.

Purification and structure elucidation of the N-acetylbacillosamine-containing polysaccharide from Bacillus licheniformis ATCC 9945.

The exopolysaccharide of Bacillus licheniformis ATCC 9945 (formerly B. subtilis ATCC 9945) contains among other glycoses 4-acetamido-2-amino-2,4,6-trideoxy-D-glucose, termed N-acetylbacillosamine (Bac2N4NAc). A similar diamino glycose, 2-acetamido-4-amino-2,4,6-trideoxy-D-glucose, was found in a surface layer (S-layer) glycoprotein preparation of Clostridium symbiosum HB25. Electron microscopic studies, however, showed that B. licheniformis ATCC 9945 is not covered with an S-layer lattice, indicating that the N-acetylbacillosamine present in that organism might be a constituent of a cell wall-associated polymer. For elucidation of the structure of the N-acetylbacillosamine-containing polysaccharide, it was purified from a trichloroacetic acid extract of B. licheniformis ATCC 9945 cells. Using different hydrolysis protocols and a hydrolysate of the S-layer glycoprotein preparation from C. symbiosum HB25 as reference, the purified polysaccharide was found to contain 2,4-diamino-2,4,6-trideoxy-glucose, 2-acetamido-2-deoxy-glucose, 2-acetamido-2-deoxy-galactose and galactose in a molar ratio of 1 : 1 : 1 : 2. One- and two-dimensional NMR spectroscopy, including 800 MHz proton magnetic resonance measurements, in combination with chemical modification and degradation experiments, revealed that the polysaccharide consists of identical pyruvylated pentasaccharide repeating units with the structure: [-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-alpha-D-GlcpNAc-(1-->3)-beta-D-Bacp2N4NAc-(1-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-beta-D-GalpNAc-(1-->](n)

Cloning of a pyruvate phosphate dikinase from Trypanosoma cruzi

We have cloned and characterised a gene that encodes a putative pyruvate phosphate dikinase (PPDK) from Trypanosoma cruzi, an enzyme that catalyses the reversible conversion of phosphoenolpyruvate to pyruvate. PPDK is absent in mammalian cells, but has been found in a wide variety of other organisms, including plants and bacteria. In T. cruzi, two genes ( PPDK1 and PPDK2) are present in a tandem array localised on a 1 Mbp chromosome. Northern and Western blot analyses indicates that PPDK is expressed as a 100-kDa protein in epimastigote, amastigote and trypomastigote forms. PPDK1 and PPDK2 encode an identical protein of 100.8 kDa with a C-terminal extension ending with the sequence AKL, a signal for glycosomal import. Both T. cruzi and T. brucei enzymes possess a 23-residue insertion, that is absent in other PPDKs. A three-dimensional alignment with the crystal structure of the enzyme from Clostridium symbiosum predicts that this insertion is located on the surface of the nucleotide-binding domain. Phylogenetic studies indicate that bacterial and protist PPDKs cluster as a separate group from those of plants. The evolutionary implications and possible role of this enzyme in T. cruzi is discussed.