Engineering self-sufficiency and broadened substrate scope into indole-nitrating cytochrome P450 TxtE

TxtE is a unique cytochrome P450 that catalyses the direct regioselective nitration of L-tryptophan in the first dedicated step in thaxtomin A biosynthesis. Highly corrosive concentrated nitric and sulphuric acids are traditionally used under harsh conditions to achieve aromatic nitration. Such reagents are difficult to handle on a large scale. Furthermore, these reactions lack regiospecificity, often resulting in a mixture of regioisomers. In contrast, TxtE catalyses regiospecific nitration of L-tryptophan at the 4-position using the artificial NO donor 2-(N,N-diethylamino)-diazenolate 2-oxide (DEANO), molecular oxygen, Fd and Fr redox partners and NADPH. The reaction proceeds at ambient temperature and the reagents are straightforward to handle, making them attractive for use on an industrial scale. Abstract figure. Nitration of L-Trp by wild-type TxtE and nitration of tryptamine by a TxtE-BM3R variant. To develop TxtE into an industrially useful biocatalyst, the enzyme was firstly fused to the reductase domain of P450BM3, a naturally occurring self-sufficient bacterial cytochrome P450 from Bacillus megaterium, eliminating the requirement for exogenous redox partner proteins. The ability of TxtE-BM3R to nitrate a range of L-Trp analogues and other aromatic compounds was then investigated, revealing TxtE is tolerant to minor substrate modifications, such as indole ring substitutions. A recently-published X-ray crystal structure of the enzyme was exploited to direct high-throughput enzyme engineering efforts focussed on improving turnover and broadening its substrate scope. Twenty-two site-saturation libraries were screened against the natural substrate and initial results revealed several mutants that improve conversion by up to 3.5 times that of the wild-type enzyme. Lastly, an R59X library was screened for tryptamine nitration, identifying two mutants capable of nitrating this biosynthetically-important molecule. Investigations into the substrate scope of this variant uncovered activity towards several molecules not accepted by the wild-type enzyme including a tricyclic compound