Catalytic promiscuity of two plant P450 enzymes: CYP725A4 from Taxus cuspidata and CYP71B102 from Isatis Tinctoria

Author

Laxmi Sagwan-Barkdoll, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2018

Degree Name

Doctor of Philosophy

Department

Plant Biology

First Advisor

Anterola, Aldwin

Abstract

Plants are abundant in cytochrome P450s constituting about 1% of their protein coding genes. Some of these P450s catalyze oxidation reactions in metabolic pathways that lead to valuable compounds, like the anticancer drug paclitaxel, the blue pigment indigo and the promising antileukemic agent indirubin. The promiscuous nature of P450 catalysis enables simultaneous production of indirubin and indigo from a common substrate, but it also decreases the yield of paclitaxel in both plants and heterologous hosts, respectively. In this thesis, the catalytic promiscuity of CYP725A4 from Taxus cuspidata and CYP71B102 from Isatis tinctoria were investigated. CYP725A4 and CYP71B102 are involved in the biosynthesis of paclitaxel and indigo/indirubin pathways, respectively. CYP725A4 is known to catalyze the hydroxylation of endotaxadiene to taxadiene-5α-ol (T5α-ol), a precursor to paclitaxel, while CYP71B102 catalyzes the production of indigo and indirubin via hydroxylation of indole. CYP725A4 exhibited catalytic promiscuity upon heterologous expression in Escherchia coli producing 5(12)-oxa-3(11)-cyclotaxane (OCT) and 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as major products, and T5α-ol as a minor product with trace amounts of unidentified monooxygenated taxanes. The presence of T5α-ol was confirmed by comparing its gas chromatography and mass spectroscopy (GC-MS) retention time and spectrum with a standard T5α-ol, while those of others were verified by matching mass spectra from previous studies. Coexpression of CYP725A4 with cytochrome P450 reductase, CPR (as either fused or separate proteins) and cytochrome b5 (Cb5) did not affect the ratios of OCT, iso-OCT, and T5α-ol, although Cb5 had an apparently negative impact on CYP725A4 activity. Attempts to modify the catalytic promiscuity of CYP725A4 were conducted by mutating key residues at the active site of the enzyme. A mutant V3741 increased the production of T5α-ol by ~10 fold, although it still supported the formation of OCT and iso-OCT as major products. The levels of these compounds in V374I were almost 45% less than in native CYP725A4. Site-directed mutagenesis was also performed on taxadiene synthase (TS) to find a mutant that only produced exotaxadiene, which could be provided to CYP725A4 as an alternative substrate. Among the TS mutants generated, none were capable of producing only exotaxadiene, but two of the TS mutants, Y684C and Q609E, produced a reasonable amount of exotaxadiene. However, coexpression of these mutants with CYP725A4 and TCPR continued to produce OCT and iso-OCT. When CYP71B102 was expressed in E. coli, indigo was the main product, while indirubin and 2-oxindole were minor products, as verified by high-performance liquid chromatography (HPLC). Half-strength terrific broth (TB) medium in combination with 5-aminolevulinic acid supplementation and isatin hydrolase coexpression altogether increased indigo formation, while supplementation with isatin and 2-oxindole increased indirubin formation. The results of this study showed that the catalytic promiscuity of CYP725A4 and CYP71B102 could be modulated by metabolic and enzyme engineering to increase the yield of commercially important compounds, like paclitaxel, indigo and indirubin.