Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus. 2004

Sedef Tunca, and Ebru I Yilmaz, and Jacqueline Piret, and Paloma Liras, and Gülay Ozcengiz
Department of Biology, Middle East Technical University, Ankara, Turkey.

Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of aspartokinase, ORF2 as the small (beta) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.

UI MeSH Term Description Entries
D009876 Operon In bacteria, a group of metabolically related genes, with a common promoter, whose transcription into a single polycistronic MESSENGER RNA is under the control of an OPERATOR REGION. Operons
D002513 Cephamycins Naturally occurring family of beta-lactam cephalosporin-type antibiotics having a 7-methoxy group and possessing marked resistance to the action of beta-lactamases from gram-positive and gram-negative organisms. Antibiotics, Cephamycin,Cephamycin,Cephamycin Antibiotics
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D001222 Aspartate Kinase An enzyme that catalyzes the formation of beta-aspartyl phosphate from aspartic acid and ATP. Threonine serves as an allosteric regulator of this enzyme to control the biosynthetic pathway from aspartic acid to threonine. EC 2.7.2.4. Aspartokinase,Aspartate Kinase I,Aspartate Kinase II,Aspartate Kinase III,Aspartyl Kinase,Kinase I, Aspartate,Kinase II, Aspartate,Kinase III, Aspartate,Kinase, Aspartate,Kinase, Aspartyl
D001223 Aspartate-Semialdehyde Dehydrogenase An enzyme that catalyzes the conversion of L-aspartate 4-semialdehyde, orthophosphate, and NADP+ to yield L-4-aspartyl phosphate and NADPH. EC 1.2.1.11. Semiaspartate Aldehyde Dehydrogenase,Aldehyde Dehydrogenase, Semiaspartate,Aspartate Semialdehyde Dehydrogenase,Dehydrogenase, Aspartate-Semialdehyde,Dehydrogenase, Semiaspartate Aldehyde
D012294 Rifamycins A group of ANTI-BACTERIAL AGENTS characterized by a chromophoric naphthohydroquinone group spanned by an aliphatic bridge not previously found in other known ANTI-BACTERIAL AGENTS. They have been isolated from fermentation broths of Streptomyces mediterranei. Rifamycin,Rifomycin,Rifomycins
D013302 Streptomyces A genus of bacteria that form a nonfragmented aerial mycelium. Many species have been identified with some being pathogenic. This genus is responsible for producing a majority of the ANTI-BACTERIAL AGENTS of practical value.
D016366 Open Reading Frames A sequence of successive nucleotide triplets that are read as CODONS specifying AMINO ACIDS and begin with an INITIATOR CODON and end with a stop codon (CODON, TERMINATOR). ORFs,Protein Coding Region,Small Open Reading Frame,Small Open Reading Frames,sORF,Unassigned Reading Frame,Unassigned Reading Frames,Unidentified Reading Frame,Coding Region, Protein,Frame, Unidentified Reading,ORF,Open Reading Frame,Protein Coding Regions,Reading Frame, Open,Reading Frame, Unassigned,Reading Frame, Unidentified,Region, Protein Coding,Unidentified Reading Frames
D017385 Sequence Homology The degree of similarity between sequences. Studies of AMINO ACID SEQUENCE HOMOLOGY and NUCLEIC ACID SEQUENCE HOMOLOGY provide useful information about the genetic relatedness of genes, gene products, and species. Homologous Sequences,Homologs, Sequence,Sequence Homologs,Homolog, Sequence,Homologies, Sequence,Homologous Sequence,Homology, Sequence,Sequence Homolog,Sequence Homologies,Sequence, Homologous,Sequences, Homologous
D017422 Sequence Analysis, DNA A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis. DNA Sequence Analysis,Sequence Determination, DNA,Analysis, DNA Sequence,DNA Sequence Determination,DNA Sequence Determinations,DNA Sequencing,Determination, DNA Sequence,Determinations, DNA Sequence,Sequence Determinations, DNA,Analyses, DNA Sequence,DNA Sequence Analyses,Sequence Analyses, DNA,Sequencing, DNA

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