13 results on '"Sim, T. S."'
Search Results
2. Thermostable malate synthase of Streptomyces thermovulgaris.
- Author
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Goh LL, Koh R, Loke P, and Sim TS
- Subjects
- Amino Acid Sequence, Cloning, Molecular, Enzyme Activation, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Malate Synthase isolation & purification, Malate Synthase metabolism, Protein Denaturation, Hot Temperature, Industrial Microbiology methods, Malate Synthase genetics, Streptomyces enzymology, Streptomyces genetics
- Abstract
The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40-60 degrees C) than those of scMS (28-37 degrees C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9-91.4% amino acid identities, with stMS possessing slightly more charged residues (approximately 31%) than its mesophilic counterparts (approximately 28-29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.
- Published
- 2003
- Full Text
- View/download PDF
3. C-terminus modification of Streptomyces clavuligerus deacetoxycephalosporin C synthase improves catalysis with an expanded substrate specificity.
- Author
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Chin HS and Sim TS
- Subjects
- Amino Acid Substitution, Biological Assay, Catalysis, Chromatography, High Pressure Liquid, Hydrophobic and Hydrophilic Interactions, Intramolecular Transferases chemistry, Mutagenesis, Site-Directed, Penicillins chemistry, Protein Structure, Tertiary, Substrate Specificity, Intramolecular Transferases genetics, Intramolecular Transferases metabolism, Penicillin-Binding Proteins, Penicillins metabolism, Streptomyces enzymology
- Abstract
The biosynthesis of cephalosporins is catalyzed by deacetoxycephalosporin C synthase (DAOCS). Based on computational, biochemical, and structural analyses, it has been proposed that modification of the C-terminus of DAOCS might be a constructive strategy for engineering improvement in enzyme activity. Therefore, five hydrophilic residues namely N301, Y302, N304, R306, and R307 located in proximity to the C-terminus of Streptomyces clavuligerus DAOCS (scDAOCS) were selected and each substituted with a hydrophobic leucine residue. Substitutions at positions 304, 306, and 307 created mutant scDAOCSs with improved efficiencies in penicillin analog conversion up to 397%. And since it has been previously advocated that the C-terminus is crucial for guiding substrate entry, a truncated mutant DAOCS was constructed to assess its involvement. The truncation of the C-terminus at position 310 in the wild-type scDAOCS resulted in reduction of indiscriminate conversion of penicillin analog but this defect was compensated by the replacement of asparagine with leucine at position 304., ((c) 2002 Elsevier Science (USA).)
- Published
- 2002
- Full Text
- View/download PDF
4. Purification and characterization of recombinant malate synthase enzymes from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585.
- Author
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Loke P, Goh LL, Seng Soh B, Yeow P, and Sim TS
- Subjects
- Chromatography, Affinity, Cloning, Molecular, DNA, Bacterial chemistry, DNA, Bacterial genetics, Escherichia coli enzymology, Escherichia coli genetics, Glutathione Transferase biosynthesis, Glutathione Transferase genetics, Glutathione Transferase isolation & purification, Kinetics, Malate Synthase biosynthesis, Malate Synthase genetics, Malate Synthase isolation & purification, Polymerase Chain Reaction, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Streptomyces genetics, Malate Synthase metabolism, Streptomyces enzymology
- Abstract
Malate synthases (MS) from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585 were cloned by polymerase chain reaction into a glutathione S-transferase (GST) fusion expression vector and heterologously expressed in Escherichia coli. The fusion GST-MS construct improved the soluble expression of MS by approximately 10-fold compared to the soluble expression of nonfusion MS. With the significant improvement in levels of soluble MS, purification and subsequent cleavage of recombinant MS from GST were facilitated in this study. Using purified enzymes, optimized parameters, which achieved maximal specific activity, were established in the enzymatic assay for streptomycete MS. The average purified specific activities of S. coelicolor and S. clavuligerus MS were 26199 and 11821 nmol/mg min, respectively. Furthermore, enzymatic analysis revealed that the two streptomycete MS displayed a similar Km value for acetyl-CoA, but S. coelicolor MS had a Km value for glyoxylate that is approximately sixfold higher than S. clavuligerus MS.
- Published
- 2002
- Full Text
- View/download PDF
5. Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585.
- Author
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Soh BS, Loke P, and Sim TS
- Subjects
- Bacterial Proteins, Cloning, Molecular, DNA chemistry, Escherichia coli genetics, Escherichia coli metabolism, Isocitrate Lyase biosynthesis, Isocitrate Lyase isolation & purification, Molecular Sequence Data, Open Reading Frames, Operon, Phylogeny, Streptomyces enzymology, Isocitrate Lyase genetics, Streptomyces genetics
- Abstract
The glyoxylate cycle comprising isocitrate lyase (ICL) and malate synthase (MS) is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, which encodes malate synthase has been previously cloned from Streptomyces clavuligerus NRRL 3585 and characterized. In this study, the aceA gene, encoding ICL from S. clavuligerus NRRL 3585, was obtained via genome walking experiments and PCR. The fully sequenced open reading frame encodes 436 amino acids with a deduced M(r) of 47.5 kDa, consistent with the observed M(r) (49-67.5 kDa) of most ICL enzymes reported so far. The cloned aceA gene was expressed in Escherichia coli BL21(lambdaDE3) cells, from which ICL was purified as a His-tagged product and its functionality demonstrated. Furthermore, the relationship between the carbon sources, growth and ICL activity in S. clavuligerus were investigated. Rapid growth was observed when the cells were cultured on 0.5% (w/v) glycerol, while delayed growth was observed when cells were grown on 0.5% (w/v) acetate. However, in both cases, high levels of ICL activity coincided with a cessation of growth, suggesting a late physiological role played by ICL in the natural host, S. clavuligerus.
- Published
- 2001
- Full Text
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6. Replacement of arginine-171 and aspartate-453 in Streptomyces coelicolor malate synthase A by site-directed mutagenesis inactivates the enzyme.
- Author
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Goh LL, Loke P, and Sim TS
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Arginine genetics, Aspartic Acid genetics, Electrophoresis, Polyacrylamide Gel, Enzyme Activation genetics, Escherichia coli genetics, Malate Synthase analysis, Malate Synthase genetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Sequence Homology, Amino Acid, Streptomyces genetics, Arginine metabolism, Aspartic Acid metabolism, Malate Synthase metabolism, Streptomyces enzymology
- Abstract
Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA. Escherichia coli is known to possess two forms of malate synthase, A and G respectively. The recent elucidation of the E. coli malate synthase G crystal structure suggested two residues, Arg338 and Asp631, are essential for catalysis. Multiple sequence alignment of 26 known malate synthase enzymes revealed that the two proposed sites are highly conserved, despite the low homologies between the two distinct forms of the enzyme (13-18%). The conservation of these residues in both forms of malate synthase suggests that they possess a similar catalytic strategy. Thus, despite the absence of a three-dimensional structure for malate synthase A, the significance of this enzyme in the primary metabolic pathway has prompted the investigation of the involvement of the corresponding residues, Arg171 and Asp453, in Streptomyces coelicolor malate synthase A by site-directed mutagenesis. Heterologous expression in E. coli followed by purification of the constructed mutant proteins, Arg171Leu and Asp453Ala, were performed and subsequent enzyme assays of the purified mutant proteins indicated a significant loss of catalytic activity, thus attesting to the need for the corresponding conserved residues to maintain malate synthase functionality.
- Published
- 2001
- Full Text
- View/download PDF
7. Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analogue conversion.
- Author
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Chin HS, Sim J, and Sim TS
- Subjects
- Amino Acid Substitution, Asparagine genetics, Biological Assay, Chromatography, High Pressure Liquid, Computational Biology, Intramolecular Transferases chemistry, Intramolecular Transferases genetics, Leucine genetics, Models, Molecular, Mutation, Penicillins chemistry, Protein Conformation, Intramolecular Transferases metabolism, Penicillin-Binding Proteins, Penicillins metabolism, Streptomyces enzymology
- Abstract
Superimposition of deacetoxycephalosporin C synthase (DAOCS) and isopenicillin N synthase (IPNS) structures revealed that R74, R160, R266 and N304 are strategically located in the catalytic cavity of Streptomyces clavuligerus DAOCS (scDAOCS) and are crucial for orchestrating different substrates. Substitutions at these sites to a hydrophobic leucine residue were expected to stabilize the hydrophobic substrate bound state. Substantial improvements in the biotransformation of penicillin G, ampicillin and amoxicillin to their respective cephalosporin moieties were observed using the N304L mutant scDAOCS. Thus, our results have demonstrated the enhancement of scDAOCS activity via critical computational analysis and site-directed mutagenesis of endogenous ligands., (Copyright 2001 Academic Press.)
- Published
- 2001
- Full Text
- View/download PDF
8. Replacement of tyrosine-197 and the corresponding tyrosine-195 to isoleucine in Cephalosporium acremonium and Streptomyces clavuligerus isopenicillin N synthase.
- Author
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Loke P and Sim TS
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Electrophoresis, Polyacrylamide Gel, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Oxidoreductases chemistry, Oxidoreductases isolation & purification, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Acremonium enzymology, Isoleucine, Oxidoreductases metabolism, Streptomyces enzymology, Tyrosine
- Abstract
Isopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.
- Published
- 2001
- Full Text
- View/download PDF
9. The invariant F283 and its strategic position in the hydrophobic cleft of Streptomyces jumonjinensis isopenicillin N synthase active site are functionally important.
- Author
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Wong E, Sim J, and Sim TS
- Subjects
- Amino Acid Sequence, Base Sequence, Catalytic Domain genetics, Conserved Sequence, DNA Primers genetics, Gene Expression, Genes, Bacterial, Intramolecular Transferases chemistry, Intramolecular Transferases genetics, Intramolecular Transferases metabolism, Models, Molecular, Mutagenesis, Site-Directed, Oxidoreductases genetics, Oxidoreductases metabolism, Phenylalanine chemistry, Protein Conformation, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Streptomyces genetics, Oxidoreductases chemistry, Penicillin-Binding Proteins, Streptomyces enzymology
- Abstract
Isopenicillin N synthase (IPNS) and related non-haem iron-binding enzymes including deacetoxycephalosporin C synthase (DAOCS) are proposed to have structurally similar active centers. Sequence alignment and computational structural analyses of predicted structures revealed 11 highly conserved hydrophobic amino acid residues in 134 IPNS-related enzymes form a contiguous hydrophobic patch in the IPNS active center, wherein F283 is strategically positioned. The investigation of single and double mutations at F283, adjacent (L284) and proximal sites (N285 and S216) of Streptomyces jumonjinensis IPNS advocate the explicit importance of the phenyl ring at position 283. A similarly placed phenylalanine (F264) in DAOCS was found to be also crucial for its enzyme activity., (Copyright 2001 Academic Press.)
- Published
- 2001
- Full Text
- View/download PDF
10. Molecular cloning, heterologous expression, and functional characterisation of a malate synthase gene from Streptomyces coelicolor A3(2).
- Author
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Loke P and Sim TS
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Cloning, Molecular, Escherichia coli enzymology, Escherichia coli genetics, Genes, Bacterial, Malate Synthase chemistry, Molecular Sequence Data, Polymerase Chain Reaction methods, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Streptomyces genetics, Malate Synthase genetics, Malate Synthase metabolism, Streptomyces enzymology
- Abstract
With the rapid generation of genetic information from the Streptomyces coelicolor genome project, deciphering the relevant gene products is critical for understanding the genetics of this model streptomycete. A putative malate synthase gene (aceB) from S. coelicolor A3(2) was identified by homology-based analysis, cloned by polymerase chain reaction, and fully sequenced on both strands. The putative malate synthase from S. coelicolor has an amino acid identity of 77% with the malate synthase of S. clavuligerus, and possesses an open reading frame which codes for a protein of 540 amino acids. In order to establish the identity of this gene, the putative aceB clones were subcloned into the expression vector pET24a, and heterologously expressed in Escherichia coli BL21(DE3). Soluble cell-free extracts containing the recombinant putative malate synthase exhibited a specific activity of 1623 (nmol.mg-1.min-1), which is an increment of 92-fold compared to the non-recombinant control. Thus, the gene product was confirmed to be a malate synthase. Interestingly, the specific activity of S. coelicolor malate synthase was found to be almost 8-fold higher than the specific activity of S. clavuligerus malate synthase under similar expression conditions. Furthermore, the genomic organisation of the three Streptomyces aceB genes cloned thus far is different from that of other bacterial malate synthases, and warrants further investigation.
- Published
- 2000
11. Mutational evidence supporting the involvement of tripartite residues His183, Asp185, and His243 in Streptomyces clavuligerus deacetoxycephalosporin C synthase for catalysis.
- Author
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Sim J and Sim TS
- Subjects
- Aspartic Acid genetics, Catalysis, Catalytic Domain, Gene Expression, Histidine genetics, Intramolecular Transferases chemistry, Intramolecular Transferases genetics, Mutagenesis, Site-Directed, Protein Structure, Secondary, Solubility, Streptomyces genetics, Streptomyces metabolism, Aspartic Acid metabolism, Histidine metabolism, Intramolecular Transferases metabolism, Penicillin-Binding Proteins, Streptomyces enzymology
- Abstract
Deacetoxycephalosporin C synthase (DAOCS) is a non-heme iron-binding and alpha-ketoglutarate dependent enzyme involved in catalyzing the biosynthesis of cephalosporins and cephamycins, antibiotics more potent than penicillins. In the crystal structure complex of Streptomyces clavuligerus DAOCS (scDAOCS), it was proposed that histidine-183, aspartate-185, and histidine-243 are putative iron-binding ligands. However, coordinates proposed for crystal structures of proteins may not definitely comply with catalysis. Hence, site-directed mutagenesis was done to replace each of these amino acid residues with leucine. The constructed expression vectors bearing the mutations were found to express the respective scDAOCS mutant enzymes at high levels in Escherichia coli BL21(DE3). Through enzymatic assays, it was shown that while the wildtype enzyme could convert penicillin to a more active cephalosporin, the substitution of the three proposed iron-binding sites of scDAOCS completely abolished the same activity in the respective mutant enzymes. Thus, these results clearly indicate that histidine-183, aspartate-185, and histidine-243 of scDAOCS are essential for the ring expansion activity.
- Published
- 2000
- Full Text
- View/download PDF
12. PCR cloning, heterologous expression, and characterization of isopenicillin N synthase from Streptomyces lipmanii NRRL 3584.
- Author
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Loke P, Ng CP, and Sim TS
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Oxidoreductases metabolism, Polymerase Chain Reaction, Sequence Alignment, Sequence Analysis, DNA, Sequence Deletion, Streptomyces enzymology, Oxidoreductases genetics, Penicillins metabolism, Streptomyces genetics
- Abstract
A key step which involves the cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to the bicyclic ring structure of isopenicillin N in the penicillin and cephalosporin biosynthetic pathway, is catalyzed by isopenicillin N synthase (IPNS). In this study, an IPNS gene from Streptomyces lipmanii NRRL 3584 (slIPNS) was cloned via PCR-based homology cloning, sequenced and expressed in Escherichia coli. Soluble slIPNS was overexpressed up to 21% of total soluble protein, and verified to be functionally active when in an IPNS enzymatic assay. Sequence comparison of the slIPNS gene obtained (excluding the consensus primer sequences) with another cloned IPNS from S. lipmanii 16884.3, revealed one three-nucleotide deletion and three closely-spaced single nucleotide deletions. Furthermore, this paper also reports the first instance of the usage of PCR as an alternative and rapid strategy for IPNS cloning using consensus primers.
- Published
- 2000
13. Glutamine-230 influences enzyme solubility but not catalysis in Streptomyces clavuligerus isopenicillin N synthase.
- Author
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Loke P and Sim TS
- Subjects
- Catalysis, Immunoblotting, Mutagenesis, Site-Directed, Solubility, Streptomyces genetics, Glutamine metabolism, Oxidoreductases chemistry, Oxidoreductases metabolism, Streptomyces enzymology
- Abstract
The conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N is dependent upon the catalytic action of isopenicillin N synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. Recent catalytic investigations on the conserved glutamine-230 in the bacterial Streptomyces jumonjinensis IPNS and the corresponding glutamine-234 in the fungal Cephalosporium acremonium IPNS showed contrasting results whereby the former was suggested to be essential for IPNS activity whereas the latter was found not to be so. In order to unravel these conflicting results, we report the site-directed mutagenesis investigation on the corresponding glutamine-230 in a third IPNS isozyme, which is the bacterial Streptomyces clavuligerus IPNS (scIPNS). IPNS enzymatic assays showed that catalytic activity of the mutant Q230L scIPNS was reduced but not eliminated. Moreover, the solubility of the mutant enzyme was also markedly reduced. Hence, we can conclude that glutamine-230 in scIPNS is not essential for catalysis and correspondingly in all IPNS.
- Published
- 1999
- Full Text
- View/download PDF
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