title: A specific cytochrome P450 hydroxylase in herboxidiene biosynthesis.
abstract: The anti-cholesterol natural product herboxidiene is synthesized by a noniterative modular polyketide synthase (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG) in Streptomyces chromofuscus A7847. In this work, the putative monooxygenase HerG was expressed in Escherichia coli and the purified enzyme was subjected to biochemical studies. It was identified as a cytochrome P450 enzyme responsible for the stereospecific hydroxylation at C-18. This enzyme is highly substrate-specific as it efficiently hydroxylates 18-deoxy-25-demethyl-herboxidiene, but showed no activity towards 18-deoxy-herboxidiene. The kcat/Km value for the HerG-catalyzed hydroxylation of 18-deoxy-25-demethyl-herboxidiene was determined to be 1669.70±47.36 M(-1) s(-1). In vitro co-reaction of HerG with the methyltransferase HerF and analysis of the product formation in S. chromofuscus A7847 revealed that the biosynthetic intermediate 18-deoxy-25-demethyl-herboxidiene is successively hydroxylated at C-18 by HerG and methylated at 17-OH to yield the final product herboxidiene. The minor metabolite 18-deoxy-hereboxidiene is a byproduct of the biosynthetic pathway.
authors.affiliation: Department of Applied Chemistry and Biological Engineering, College of Chemical Engineering, Northeast Dianli University, Jilin, Jilin 132012, China; Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322, United States. Electronic address: yudy@mail.nedu.edu.cn.
authors.forename: Dayu
authors.initials: D
authors.lastname: Yu
authors.affiliation: Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322, United States.
authors.forename: Fuchao
authors.initials: F
authors.lastname: Xu
authors.affiliation: Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322, United States.
authors.forename: Lei
authors.initials: L
authors.lastname: Shao
authors.affiliation: Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322, United States. Electronic address: jixun.zhan@usu.edu.
authors.forename: Jixun
authors.initials: J
authors.lastname: Zhan
mesh_terms: D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005233:Fatty Alcohols / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011714:Pyrans / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D005233:Fatty Alcohols; D011714:Pyrans; C076087:herboxidiene; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases
keywords: Cytochrome P450; Herboxidiene; Hydroxylation; Polyketide biosynthesis; Specificity