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Structure, function and substrate tolerance of TxtC
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Alkhalaf, Lona M. (2013) Structure, function and substrate tolerance of TxtC. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2717011~S1
Abstract
Thaxtomins are a family of phytotoxins produced by Streptomyces scabies and other
Streptomyces species, which cause scab disease in root crops, such as potato, carrot and radish.
The final step of their biosynthesis involves sequential hydroxylation reactions catalysed by the
cytochrome P450 (CYP), TxtC (figure 1). This CYP is of particular interest because it is
chemically challenging to selectively hydroxylate at the phenylalanine alpha-position, and
thaxtomins have been shown to have herbicidal activity. The hydroxyl groups introduced by
TxtC have been shown to contribute both to the phytotoxicity, and solubility, of thaxtomin A.
The first target was to complete a total chemoenzymatic synthesis of thaxtomin A. Peptide
coupling of Boc-4-nitroTrp and N-Me-Phe methyl ester, cyclisation to give the monomethylated
diketopiperazine and subsequent methylation of the 4-nitroTrp amide nitrogen led to thaxtomin
D. TxtC was then utilised to complete the final two hydroxylation steps to give thaxtomin A as
a single enantiomer.
A range of TxtC substrate analogues were then synthesised in order to probe the SAR of TxtC.
Reactivity was assessed both in vitro and in vivo and indicated a fairly broad substrate tolerance,
with modification to the indole, phenyl group and N-methyl groups all tolerated to give
monohydroxylated, and in some cases dihydroxylated, products. Selectivity was comparable to
the natural substrate in those analogues with modification to the indole or N-methyl groups. A
number of the hydroxylated products have been characterised by 1H and 13C NMR which show
the major site of hydroxylation is as observed in thaxtomin A.
X-ray crystallography of TxtC with both substrates, thaxtomin D and thaxtomin B, led to
production of structures to 2.8Å and 1.7Å, respectively. These structures, as well as subsequent
docking simulations, have given insights into the SAR observed, and the mechanism by which
hydroxylation occurs.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QD Chemistry Q Science > QR Microbiology |
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Library of Congress Subject Headings (LCSH): | Toxins, Streptomyces scabies, Toxins -- Structure-activity relationships, Biosynthesis, Substrates, Hydroxylation, X-ray crystallography | ||||
Official Date: | January 2013 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Chemistry | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Challis, Gregory L. | ||||
Sponsors: | Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC); Syngenta Foundation for Sustainable Agriculture | ||||
Extent: | 1 volume (various pagings) : illustrations. | ||||
Language: | eng |
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