Molecular Cloning of the Actinomycin Synthetase Gene Cluster from Streptomyces chrysomallus and Functional Heterologous Expression of the Gene Encoding Actinomycin Synthetase II
AUTOR(ES)
Schauwecker, Florian
FONTE
American Society for Microbiology
RESUMO
The actinomycin synthetases ACMS I, II, and III catalyze the assembly of the acyl peptide lactone precursor of actinomycin by a nonribosomal mechanism. We have cloned the genes of ACMS I (acmA) and ACMS II (acmB) by hybridization screening of a cosmid library of Streptomyces chrysomallus DNA with synthetic oligonucleotides derived from peptide sequences of the two enzymes. Their genes were found to be closely linked and are arranged in opposite orientations. Hybridization mapping and partial sequence analyses indicate that the gene of an additional peptide synthetase, most likely the gene of ACMS III (acmC), is located immediately downstream of acmB in the same orientation. The protein sequence of ACMS II, deduced from acmB, shows that the enzyme contains two amino acid activation domains, which are characteristic of peptide synthetases, and an additional epimerization domain. Heterologous expression of acmB from the mel promoter of plasmid PIJ702 in Streptomyces lividans yielded a functional 280-kDa peptide synthetase which activates threonine and valine as enzyme-bound thioesters. It also catalyzes the dipeptide formation of threonyl–l-valine, which is epimerized to threonyl–d-valine. Both of these dipeptides are enzyme bound as thioesters. This catalytic activity is identical to the in vitro activity of ACMS II from S. chrysomallus.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=107190Documentos Relacionados
- Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736
- Molecular Analysis of a Gene Encoding a Cell-Bound Esterase from Streptomyces chrysomallus
- Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata
- Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from Streptomyces roseosporus
- Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase.