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Jui Shen Chiao - One of the best experts on this subject based on the ideXlab platform.
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expression in escherichia coli purification and kinetic analysis of the aspartokinase and Aspartate Semialdehyde Dehydrogenase from the rifamycin sv producing amycolatopsis mediterranei u32
Applied Microbiology and Biotechnology, 2000Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:The operon encoding aspartokinase and Aspartate Semialdehyde Dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask−) and E. coli X6118 (asd −), respectively. The A. mediterranei U32 aspartokinase and Aspartate Semialdehyde Dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and Aspartate Semialdehyde Dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and Aspartate Semialdehyde Dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for Aspartate and 2.3 mM for ATP, while Aspartate Semialdehyde Dehydrogenase has Km values of 1.25 mM for dl-Aspartate Semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by l-threonine, l-lysine, and l-methionine, but not by l-isoleucine and diaminopimelate. Aspartate Semialdehyde Dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that aspartokinase was subject to allosteric control by l-threonine. Repression of both aspartokinase and Aspartate Semialdehyde Dehydrogenase gene transcription in A. mediterranei U32 by l-lysine, l-methionine, l-threonine, and l-isoleucine were found. The network of regulation of aspartokinase and Aspartate Semialdehyde Dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.
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sequence analysis and expression of the aspartokinase and Aspartate Semialdehyde Dehydrogenase operon from rifamycin sv producing amycolatopsis mediterranei
Gene, 1999Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:Abstract A ∼4.8 kb KpnI fragment, from the upstream region of the methylmalonyl-CoA mutase gene (mutAB) of rifamycin SV-producing Amycolatopsis mediterranei, was cloned and partially sequenced. Codon preference analysis showed three complete ORFs. ORF2 is internal to ORF1, and encodes a polypeptide corresponding to 172 amino acids, whereas ORF1 encodes a polypeptide of 421 amino acids. They were identified as the encoding genes of aspartokinase α- and β-subunits by comparing the amino acid sequences with those in the database. The downstream ORF3, whose start codon was overlapped with the stop codon of both ORF1 and ORF2 by 1 bp, was identified as the Aspartate Semialdehyde Dehydrogenase gene (asd), encoding a polypeptide of 346 amino acids. Subclones containing either the ask gene or the asd gene were constructed, in which the genes could be expressed under Lac promoters. Two subclones could transform E. coli CGSC 5074 (ask-) and E. coli X6118 (asd-) to prototrophy, supporting the functional assignments. Southern hybridisation indicated that the ∼4.8 kb sequenced region represented a continuous segment in the A. mediterranei chromosome. It is concluded that ask and asd genes are present in an operon in A. mediterranei, and therefore that organisation of these two genes is the same as in most gram-positive bacteria, such as Mycobacteria, Corynebacterium glutamicum and Bacillus subtilis, but is different from Streptomyces akiyoshiensis.
Ronald E Viola - One of the best experts on this subject based on the ideXlab platform.
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Aspartate Semialdehyde Dehydrogenase inhibition suppresses the growth of the pathogenic fungus candida albicans
Drug Development Research, 2020Co-Authors: Gopal P Dahal, Dylan Launder, Katherine M M Mckeone, Joseph P Hunter, Heather R Conti, Ronald E ViolaAbstract:Potent inhibitors of an essential microbial enzyme have been shown to be effective growth inhibitors of Candida albicans, a pathogenic fungus. C. albicans is the main cause of oropharyngeal candidiasis, and also causes invasive fungal infections, including systemic sepsis, leading to serious complications in immunocompromised patients. As the rates of drug-resistant fungal infections continue to rise novel antifungal treatments are desperately needed. The enzyme Aspartate Semialdehyde Dehydrogenase (ASADH) is critical for the functioning of the Aspartate biosynthetic pathway in microbes and plants. Because the Aspartate pathway is absent in humans, ASADH has the potential to be a promising new target for antifungal research. Deleting the asd gene encoding for ASADH significantly decreases the survival of C. albicans, establishing this enzyme as essential for this organism. Previously developed ASADH inhibitors were tested against several strains of C. albicans to measure their possible therapeutic impact. The more potent inhibitors show a good correlation between enzyme inhibitor potency and fungal growth inhibition. Growth curves generated by incubating different C. albicans strains with varying enzyme inhibitor levels show significant slowing of fungal growth by these inhibitors against each of these strains, similar to the effect observed with a clinical antifungal drug. The most effective inhibitors also demonstrated relatively low cytotoxicity against a human epithelial cell line. Taken together, these results establish that the ASADH enzyme is a promising new target for further development as a novel antifungal treatment against C. albicans and related fungal species.
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structural insights into inhibitor binding to a fungal ortholog of Aspartate Semialdehyde Dehydrogenase
Biochemical and Biophysical Research Communications, 2018Co-Authors: Gopal P Dahal, Ronald E ViolaAbstract:The Aspartate pathway, uniquely found in plants and microorganisms, offers novel potential targets for the development of new antimicrobial drugs. Aspartate Semialdehyde Dehydrogenase (ASADH) catalyzes production of a key intermediate at the first branch point in this pathway. Several fungal ASADH structures have been determined, but the prior crystallization conditions had precluded complex formation with enzyme inhibitors. The first inhibitor-bound and cofactor-bound structures of ASADH from the pathogenic fungi Blastomyces dermatitidis have now been determined, along with a structural and functional comparison to other ASADH family members. The structure of this new ASADH is similar to the other fungal orthologs, but with some critical differences in the orientation of some active site functional groups and in the subunit interface region. The presence of this bound inhibitor reveals the first details about inhibitor binding interactions, and the flexible orientation of its aromatic ring provides helpful insights into the design of potentially more potent and selective antifungal compounds.
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structure of a fungal form of Aspartate Semialdehyde Dehydrogenase from aspergillus fumigatus
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2017Co-Authors: Gopal P Dahal, Ronald E ViolaAbstract:Aspartate-Semialdehyde Dehydrogenase (ASADH) functions at a critical junction in the Aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of β-aspartyl phosphate to produce the key intermediate Aspartate Semialdehyde. The absence of this entire pathway in humans and other mammals will allow the selective targeting of pathogenic microorganisms for antimicrobial development. Here, the X-ray structure of a new form of ASADH from the pathogenic fungal species Aspergillus fumigatus has been determined. The overall structure of this enzyme is similar to those of its bacterial orthologs, but there are some critical differences both in biological assembly and in secondary-structural features that can potentially be exploited for the development of species-selective drugs with selective toxicity against infectious fungal organisms.
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structure of a fungal form of Aspartate Semialdehyde Dehydrogenase from cryptococcus neoformans
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2015Co-Authors: Gopal P Dahal, Ronald E ViolaAbstract:Aspartate Semialdehyde Dehydrogenase (ASADH) functions at a critical junction in the Aspartate-biosynthetic pathway and represents a valid target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of β-aspartyl phosphate to produce the key intermediate Aspartate Semialdehyde. Production of this intermediate represents the first committed step in the biosynthesis of the essential amino acids methionine, isoleucine and threonine in fungi, and also the amino acid lysine in bacteria. The structure of a new fungal form of ASADH from Cryptococcus neoformans has been determined to 2.6 A resolution. The overall structure of CnASADH is similar to those of its bacterial orthologs, but with some critical differences both in biological assembly and in secondary-structural features that can potentially be exploited for the development of species-selective drugs.
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elaboration of a fragment library hit produces potent and selective Aspartate Semialdehyde Dehydrogenase inhibitors
Bioorganic & Medicinal Chemistry, 2015Co-Authors: Bharani Thangavelu, Pravin Bhansali, Ronald E ViolaAbstract:Aspartate-β-Semialdehyde Dehydrogenase (ASADH) lies at the first branch point in the Aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme.
Weiwen Zhang - One of the best experts on this subject based on the ideXlab platform.
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expression in escherichia coli purification and kinetic analysis of the aspartokinase and Aspartate Semialdehyde Dehydrogenase from the rifamycin sv producing amycolatopsis mediterranei u32
Applied Microbiology and Biotechnology, 2000Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:The operon encoding aspartokinase and Aspartate Semialdehyde Dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask−) and E. coli X6118 (asd −), respectively. The A. mediterranei U32 aspartokinase and Aspartate Semialdehyde Dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and Aspartate Semialdehyde Dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and Aspartate Semialdehyde Dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for Aspartate and 2.3 mM for ATP, while Aspartate Semialdehyde Dehydrogenase has Km values of 1.25 mM for dl-Aspartate Semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by l-threonine, l-lysine, and l-methionine, but not by l-isoleucine and diaminopimelate. Aspartate Semialdehyde Dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that aspartokinase was subject to allosteric control by l-threonine. Repression of both aspartokinase and Aspartate Semialdehyde Dehydrogenase gene transcription in A. mediterranei U32 by l-lysine, l-methionine, l-threonine, and l-isoleucine were found. The network of regulation of aspartokinase and Aspartate Semialdehyde Dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.
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sequence analysis and expression of the aspartokinase and Aspartate Semialdehyde Dehydrogenase operon from rifamycin sv producing amycolatopsis mediterranei
Gene, 1999Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:Abstract A ∼4.8 kb KpnI fragment, from the upstream region of the methylmalonyl-CoA mutase gene (mutAB) of rifamycin SV-producing Amycolatopsis mediterranei, was cloned and partially sequenced. Codon preference analysis showed three complete ORFs. ORF2 is internal to ORF1, and encodes a polypeptide corresponding to 172 amino acids, whereas ORF1 encodes a polypeptide of 421 amino acids. They were identified as the encoding genes of aspartokinase α- and β-subunits by comparing the amino acid sequences with those in the database. The downstream ORF3, whose start codon was overlapped with the stop codon of both ORF1 and ORF2 by 1 bp, was identified as the Aspartate Semialdehyde Dehydrogenase gene (asd), encoding a polypeptide of 346 amino acids. Subclones containing either the ask gene or the asd gene were constructed, in which the genes could be expressed under Lac promoters. Two subclones could transform E. coli CGSC 5074 (ask-) and E. coli X6118 (asd-) to prototrophy, supporting the functional assignments. Southern hybridisation indicated that the ∼4.8 kb sequenced region represented a continuous segment in the A. mediterranei chromosome. It is concluded that ask and asd genes are present in an operon in A. mediterranei, and therefore that organisation of these two genes is the same as in most gram-positive bacteria, such as Mycobacteria, Corynebacterium glutamicum and Bacillus subtilis, but is different from Streptomyces akiyoshiensis.
Yunliu Yang - One of the best experts on this subject based on the ideXlab platform.
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expression in escherichia coli purification and kinetic analysis of the aspartokinase and Aspartate Semialdehyde Dehydrogenase from the rifamycin sv producing amycolatopsis mediterranei u32
Applied Microbiology and Biotechnology, 2000Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:The operon encoding aspartokinase and Aspartate Semialdehyde Dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask−) and E. coli X6118 (asd −), respectively. The A. mediterranei U32 aspartokinase and Aspartate Semialdehyde Dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and Aspartate Semialdehyde Dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and Aspartate Semialdehyde Dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for Aspartate and 2.3 mM for ATP, while Aspartate Semialdehyde Dehydrogenase has Km values of 1.25 mM for dl-Aspartate Semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by l-threonine, l-lysine, and l-methionine, but not by l-isoleucine and diaminopimelate. Aspartate Semialdehyde Dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that aspartokinase was subject to allosteric control by l-threonine. Repression of both aspartokinase and Aspartate Semialdehyde Dehydrogenase gene transcription in A. mediterranei U32 by l-lysine, l-methionine, l-threonine, and l-isoleucine were found. The network of regulation of aspartokinase and Aspartate Semialdehyde Dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.
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sequence analysis and expression of the aspartokinase and Aspartate Semialdehyde Dehydrogenase operon from rifamycin sv producing amycolatopsis mediterranei
Gene, 1999Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:Abstract A ∼4.8 kb KpnI fragment, from the upstream region of the methylmalonyl-CoA mutase gene (mutAB) of rifamycin SV-producing Amycolatopsis mediterranei, was cloned and partially sequenced. Codon preference analysis showed three complete ORFs. ORF2 is internal to ORF1, and encodes a polypeptide corresponding to 172 amino acids, whereas ORF1 encodes a polypeptide of 421 amino acids. They were identified as the encoding genes of aspartokinase α- and β-subunits by comparing the amino acid sequences with those in the database. The downstream ORF3, whose start codon was overlapped with the stop codon of both ORF1 and ORF2 by 1 bp, was identified as the Aspartate Semialdehyde Dehydrogenase gene (asd), encoding a polypeptide of 346 amino acids. Subclones containing either the ask gene or the asd gene were constructed, in which the genes could be expressed under Lac promoters. Two subclones could transform E. coli CGSC 5074 (ask-) and E. coli X6118 (asd-) to prototrophy, supporting the functional assignments. Southern hybridisation indicated that the ∼4.8 kb sequenced region represented a continuous segment in the A. mediterranei chromosome. It is concluded that ask and asd genes are present in an operon in A. mediterranei, and therefore that organisation of these two genes is the same as in most gram-positive bacteria, such as Mycobacteria, Corynebacterium glutamicum and Bacillus subtilis, but is different from Streptomyces akiyoshiensis.
Guoping Zhao - One of the best experts on this subject based on the ideXlab platform.
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expression in escherichia coli purification and kinetic analysis of the aspartokinase and Aspartate Semialdehyde Dehydrogenase from the rifamycin sv producing amycolatopsis mediterranei u32
Applied Microbiology and Biotechnology, 2000Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:The operon encoding aspartokinase and Aspartate Semialdehyde Dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask−) and E. coli X6118 (asd −), respectively. The A. mediterranei U32 aspartokinase and Aspartate Semialdehyde Dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and Aspartate Semialdehyde Dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and Aspartate Semialdehyde Dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for Aspartate and 2.3 mM for ATP, while Aspartate Semialdehyde Dehydrogenase has Km values of 1.25 mM for dl-Aspartate Semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by l-threonine, l-lysine, and l-methionine, but not by l-isoleucine and diaminopimelate. Aspartate Semialdehyde Dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that aspartokinase was subject to allosteric control by l-threonine. Repression of both aspartokinase and Aspartate Semialdehyde Dehydrogenase gene transcription in A. mediterranei U32 by l-lysine, l-methionine, l-threonine, and l-isoleucine were found. The network of regulation of aspartokinase and Aspartate Semialdehyde Dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.
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sequence analysis and expression of the aspartokinase and Aspartate Semialdehyde Dehydrogenase operon from rifamycin sv producing amycolatopsis mediterranei
Gene, 1999Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:Abstract A ∼4.8 kb KpnI fragment, from the upstream region of the methylmalonyl-CoA mutase gene (mutAB) of rifamycin SV-producing Amycolatopsis mediterranei, was cloned and partially sequenced. Codon preference analysis showed three complete ORFs. ORF2 is internal to ORF1, and encodes a polypeptide corresponding to 172 amino acids, whereas ORF1 encodes a polypeptide of 421 amino acids. They were identified as the encoding genes of aspartokinase α- and β-subunits by comparing the amino acid sequences with those in the database. The downstream ORF3, whose start codon was overlapped with the stop codon of both ORF1 and ORF2 by 1 bp, was identified as the Aspartate Semialdehyde Dehydrogenase gene (asd), encoding a polypeptide of 346 amino acids. Subclones containing either the ask gene or the asd gene were constructed, in which the genes could be expressed under Lac promoters. Two subclones could transform E. coli CGSC 5074 (ask-) and E. coli X6118 (asd-) to prototrophy, supporting the functional assignments. Southern hybridisation indicated that the ∼4.8 kb sequenced region represented a continuous segment in the A. mediterranei chromosome. It is concluded that ask and asd genes are present in an operon in A. mediterranei, and therefore that organisation of these two genes is the same as in most gram-positive bacteria, such as Mycobacteria, Corynebacterium glutamicum and Bacillus subtilis, but is different from Streptomyces akiyoshiensis.
