|Healy, F - CORNELL UNIVERSITY|
|Krasnoff, S - CORNELL UNIVERSITY|
|Wach, M - CORNELL UNIVERSITY|
|Loria, R - CORNELL UNIVERSITY|
Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 28, 2001
Publication Date: April 1, 2002
Interpretive Summary: Streptomycetes are soilborne bacteria that produce the vast majority of commercially important antibiotics, enzymes, and other compounds. Among the hundreds of described Streptomyces species, only four species are plant pathogens (S. scabies, S. acidiscabies, S. turgidiscabies and S. ipomoeae), and they are the causal agents of scab diseases of economically important root and tuber crops, such as potatoes. These organisms produce a small chemical compound called thaxtomin which causes disease symptoms of scab on susceptible crops. This work was undertaken to identify the structural genes that produce thaxtomin. One of the genes responsible for a specific biosynthetic step in the pathway was described, and the order of biosynthetic pathway has been deduced. With the identification of the genes responsible for thaxtomin biosynthesis, it will now be possible to more precisely assess the importance of this compound in causing plant disease.
Technical Abstract: The biosynthesis of the dipeptide thaxtomin phytotoxin family proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification & cyclization of two amino acid substrates is catalyzed by the txtAB-encoded thaxtomin synthetase genes. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an ORF encoding a putative P-450 monooxygenase. It was proposed that one or both of the thaxtomin A phenylalanyl hydroxyl groups are provided by the monooxygenase. S. acidiscabies 84.104 monooxygenase mutants were created using an integrative gene disruption construct & culture filtrate extracts of the mutants were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed phase HPLC anaylsis of mutant extracts revealed a prominent peak of lesser polarity than thaxtomin A, & MS data suggested the compound to be the 14, 20-didehydroxy derivative. Proton NMR assignments of the purified compound confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl). These data demonstrate that thaxtomin A biosynthesis is initiated through the condensation of L-4-nitrotryptophan & L-phenylalanine substrate amino acids, & the monooxygenase gene, txtC, is required for hydroxylation of the cyclic dipeptide. It is not known whether the monooxygenase catalyzes both oxygenations, or if the absence of a monohydryoxylated intermediate results in the premature termination of thaxtomin A biosynthesis. This second possibility would suggest that another as yet unidentified monooxygenase carries out the terminal step in thaxtomin A production. In vitro studies with the TxtC monooxygenase & the didehydroxy derivative will allow us to elucidate the role of TxtC.