TY - JOUR
T1 - Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450 CYP725A4
AU - Biggs, Bradley Walters
AU - Rouck, John Edward
AU - Kambalyal, Amogh
AU - Arnold, William
AU - Lim, Chin Giaw
AU - De Mey, Marjan
AU - Oneil-Johnson, Mark
AU - Starks, Courtney M.
AU - Das, Aditi
AU - Ajikumar, Parayil Kumaran
N1 - Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/5/20
Y1 - 2016/5/20
N2 - Natural product metabolic engineering potentially offers sustainable and affordable access to numerous valuable molecules. However, challenges in characterizing and assembling complex biosynthetic pathways have prevented more rapid progress in this field. The anticancer agent Taxol represents an excellent case study. Assembly of a biosynthetic pathway for Taxol has long been stalled at its first functionalization, putatively an oxygenation performed by the cytochrome P450 CYP725A4, due to confounding characterizations. Here, through combined in vivo (Escherichia coli), in vitro (lipid nanodisc), and metabolite stability assays, we verify the presence and likely cause of this enzymes inherent promiscuity. Thereby, we remove the possibility that promiscuity simply existed as an artifact of previous metabolic engineering approaches. Further, spontaneous rearrangement and the stabilizing effect of a hydrophobic overlay suggest a potential role for nonenzymatic chemistry in Taxols biosynthesis. Taken together, this work confirms taxadiene-5α-ol as a primary enzymatic product of CYP725A4 and provides direction for future Taxol metabolic and protein engineering efforts.
AB - Natural product metabolic engineering potentially offers sustainable and affordable access to numerous valuable molecules. However, challenges in characterizing and assembling complex biosynthetic pathways have prevented more rapid progress in this field. The anticancer agent Taxol represents an excellent case study. Assembly of a biosynthetic pathway for Taxol has long been stalled at its first functionalization, putatively an oxygenation performed by the cytochrome P450 CYP725A4, due to confounding characterizations. Here, through combined in vivo (Escherichia coli), in vitro (lipid nanodisc), and metabolite stability assays, we verify the presence and likely cause of this enzymes inherent promiscuity. Thereby, we remove the possibility that promiscuity simply existed as an artifact of previous metabolic engineering approaches. Further, spontaneous rearrangement and the stabilizing effect of a hydrophobic overlay suggest a potential role for nonenzymatic chemistry in Taxols biosynthesis. Taken together, this work confirms taxadiene-5α-ol as a primary enzymatic product of CYP725A4 and provides direction for future Taxol metabolic and protein engineering efforts.
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U2 - 10.1021/acschembio.5b00968
DO - 10.1021/acschembio.5b00968
M3 - Article
C2 - 26930136
AN - SCOPUS:84971241591
SN - 1554-8929
VL - 11
SP - 1445
EP - 1451
JO - ACS chemical biology
JF - ACS chemical biology
IS - 5
ER -