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Purification and properties of threonine aldolase from Clostridium pasteurianum.

ABSTRACT: 1. Threonine aldolase was purified about 200-fold in 10% yield from Clostridium pasteurianum and its properties were examined. The final preparation gave three bands after ionophoresis on polyacrylamide gel. 2. The purified enzyme was shown to produce glycine and acetaldehyde in stoicheiometric amounts from threonine. The reverse reaction was demonstrated qualitatively. 3. The enzyme has a broad pH optimum at 6.5-7.0. 4. The enzyme is highly specific for l-threonine. 5. The enzyme is completely inhibited by 1mm concentrations of hydroxylamine and semicarbazide. Activity is decreased to 20% of the original by treatment with cysteine plus mercaptoethanol; most of the loss is regained on incubation with pyridoxal phosphate. It is concluded that pyridoxal phosphate is a prosthetic group. 6. The relationship between velocity and substrate concentration is atypical but indicates a K(m) value of 0.42mm. 7. The enzyme was demonstrated in several other strictly anaerobic bacteria.

SUBMITTER: Dainty RH

PROVIDER: S-EPMC1178963 | biostudies-other | 1970 Apr

REPOSITORIES: biostudies-other

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Clostridium pasteurianum F1Fo ATP synthase: operon, composition, and some properties.

Project description:The atp operon encoding F1Fo ATP synthase in the fermentative obligate anaerobic bacterium Clostridium pasteurianum was sequenced. It consisted of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(delta), atpA(alpha), atpG(gamma), atpD(beta), and atpC(epsilon), which was identical to that found in many bacteria. Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes. The amount of ATPase activity in the membranes of C. pasteurianum was low compared to what has been found in many other bacteria. The F1Fo complexes solubilized from membranes of C. pasteurianum and Escherichia coli had similar masses, suggesting similar compositions for the F1Fo complexes from the two bacteria. Western blotting experiments with antibodies raised against the purified subunits of F1Fo detected the presence of eight subunits, alpha, beta, gamma, delta, epsilon, a, b, and c, in the F1Fo complex from C. pasteurianum. The F1Fo complex from C. pasteurianum was activated by thiocyanate, cyanate, or sulfhydryl compounds; inhibited by sulfite, bisulfite, or bicarbonate; and had tolerance to inhibition by dicyclohexylcarbodiimide. The target of thiol activation of the F1Fo complex from C. pasteurianum was F1. Thiocyanate and sulfite were noncompetitive with respect to substrate Mg ATP but competitive with respect to each other. The F1 and Fo parts of the F1Fo complexes from C. pasteurianum and E. coli bound to each other, but the hybrid F1Fo complexes were not functionally active.

| S-EPMC193768 | biostudies-literature

Mobilization of granulose in Clostridium pasteurianum. Purification and properties of granulose phosphorylase.

Project description:1. The granulose of Clostridium pasteurianum ATCC 6013 is degraded when the organism is incubated in a medium containing no utilizable source of carbon and energy. 2. Mobilization of the polyglucan does not occur in the presence of exogenous glucose. 3. Breakdown of granulose is effected by a constitutively synthesized alpha-1,4-polyglucan phosphorylase. 4. Partial (530-fold) purification of this granulose phosphorylase was facilitated by its being loosely bound to the native granules of its substrate polyglucan. 5. The enzyme (pH optimum 6.4) was assayed both (a) in the degradative direction, K(m) for P(i)=2.2mm, and (b) in the synthetic direction, K(m) for glucose 1-phosphate=0.05mm. No requirement for bivalent cations was evidenced. 6. Granulose phosphorylase was inhibited by various nucleotide sugars; GDP-glucose, ADP-glucose (K(i)=20mum) and UDP-glucose (K(i)=60mum) were particularly potent competitive inhibitors. ATP, NADP(+) and NADPH (at 1mm) were less effective inhibitors, whereas AMP was slightly stimulatory. 7. It would appear that granulose mobilization is favoured under conditions of low adenylate energy charge, but is prevented under conditions of ;glucose excess' chiefly by ADP-glucose-mediated inhibition of granulose phosphorylase.

| S-EPMC1168529 | biostudies-other

Clostridium pasteurianum

Project description:Nitrogen-fixing soil bacterium

| PRJNA35233 | ENA

Clostridium pasteurianum

Project description:Clostridium pasteurianum Genome sequencing

| PRJNA299496 | ENA

Closed Genome Sequence of Clostridium pasteurianum ATCC 6013.

Project description:We report here the closed genome of Clostridium pasteurianum ATCC 6013, a saccharolytic, nitrogen-fixing, and spore-forming Gram-positive obligate anaerobe. The organism is of biotechnological interest due to the production of solvents (butanol and 1,3-propanediol) but can be associated with food spoilage. The genome comprises a total of 4,351,223 bp.

| S-EPMC4335343 | biostudies-literature

Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii.

Project description:Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg-1 was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.

| S-EPMC106640 | biostudies-literature

Mechanism of proton transfer in [FeFe]-hydrogenase from Clostridium pasteurianum.

Project description:[FeFe]-Hydrogenases are complex metalloproteins that catalyze the reversible reduction of protons to molecular hydrogen utilizing a unique diiron subcluster bridged to a [4Fe4S] subcluster. Extensive studies have concentrated on the nature and catalytic activity of the active site, yet relatively little information is available concerning the mechanism of proton transport that is required for this activity. Previously, structural characterization of [FeFe]-hydrogenase from Clostridium pasteurianum indicated a potential proton transport pathway involving four residues (Cys-299, Glu-279, Ser-319, and Glu-282) that connect the active site to the enzyme surface. Here, we demonstrate that substitution of any of these residues resulted in a drastic reduction in hydrogenase activity relative to the native enzyme, supporting the importance of these residues in catalysis. Inhibition studies of native and amino acid-substituted enzymes revealed that Zn(2+) specifically blocked proton transfer by binding to Glu-282, confirming the role of this residue in the identified pathway. In addition, all four of these residues are strictly conserved, suggesting that they may form a proton transport pathway that is common to all [FeFe]-hydrogenases.

| S-EPMC3207428 | biostudies-literature

Gene cloning, nucleotide sequencing, and purification and characterization of the low-specificity L-threonine aldolase from Pseudomonas sp. strain NCIMB 10558.

Project description:A low-specificity L-threonine aldolase (L-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5'-phosphate as a coenzyme, is strictly L specific at the alpha position, whereas it cannot distinguish between threo and erythro forms at the beta position. In addition to threonine, the enzyme also acts on various other L-beta-hydroxy-alpha-amino acids, including L-beta-3,4-dihydroxyphenylserine, L-beta-3,4-methylenedioxyphenylserine, and L-beta-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity L-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the L-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5'-phosphate of the enzyme to catalyze the reversible aldol reaction.

| S-EPMC106081 | biostudies-literature

Clostridium pasteurianum BC1

Project description:Nitrogen-fixing bacterium and potential bioremediator.

| PRJNA80 | ENA

Towards improved butanol production through targeted genetic modification of Clostridium pasteurianum.

Project description:Declining fossil fuel reserves, coupled with environmental concerns over their continued extraction and exploitation have led to strenuous efforts to identify renewable routes to energy and fuels. One attractive option is to convert glycerol, a by-product of the biodiesel industry, into n-butanol, an industrially important chemical and potential liquid transportation fuel, using Clostridium pasteurianum. Under certain growth conditions this Clostridium species has been shown to predominantly produce n-butanol, together with ethanol and 1,3-propanediol, when grown on glycerol. Further increases in the yields of n-butanol produced by C. pasteurianum could be accomplished through rational metabolic engineering of the strain. Accordingly, in the current report we have developed and exemplified a robust tool kit for the metabolic engineering of C. pasteurianum and used the system to make the first reported in-frame deletion mutants of pivotal genes involved in solvent production, namely hydA (hydrogenase), rex (Redox response regulator) and dhaBCE (glycerol dehydratase). We were, for the first time in C. pasteurianum, able to eliminate 1,3-propanediol synthesis and demonstrate its production was essential for growth on glycerol as a carbon source. Inactivation of both rex and hydA resulted in increased n-butanol titres, representing the first steps towards improving the utilisation of C. pasteurianum as a chassis for the industrial production of this important chemical.

| S-EPMC5367854 | biostudies-literature

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