Structure, function and substrate tolerance of TxtC

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