Involvement of a Cytochrome P450 Monooxygenase in Thaxtomin A Biosynthesis by Streptomyces acidiscabies
Autor: | Rosemary Loria, Michael J. Wach, Frank Healy, Donna M. Gibson, Stuart B. Krasnoff |
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Rok vydání: | 2002 |
Předmět: |
DNA
Bacterial Models Molecular Indoles Cytochrome Molecular Sequence Data Hydroxylation Microbiology Streptomyces Catalysis Piperazines chemistry.chemical_compound Plant Microbiology Cytochrome P-450 Enzyme System Biosynthesis Escherichia coli Amino Acid Sequence Molecular Biology Sequence Homology Amino Acid biology Cytochrome P450 Monooxygenase biology.organism_classification Open reading frame chemistry Biochemistry Streptomyces acidiscabies biology.protein |
Zdroj: | Journal of Bacteriology. 184:2019-2029 |
ISSN: | 1098-5530 0021-9193 |
Popis: | The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N -methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB -encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3′ of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as 1 H- and 13 C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15- N -dimethylcyclo-( l -4-nitrotryptophyl- l -phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC , was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide. |
Databáze: | OpenAIRE |
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