title: Analysis of the cryptophycin P450 epoxidase reveals substrate tolerance and cooperativity.
abstract: Cryptophycins are potent anticancer agents isolated from Nostoc sp. ATCC 53789 and Nostoc sp. GSV 224. The most potent natural cryptophycin analogues retain a beta-epoxide at the C2'-C3' position of the molecule. A P450 epoxidase encoded by c rpE recently identified from the cryptophycin gene cluster was shown to install this key functional group into cryptophycin-4 (Cr-4) to produce cryptophycin-2 (Cr-2) in a regio- and stereospecific manner. Here we report a detailed characterization of the CrpE epoxidase using an engineered maltose binding protein (MBP)-CrpE fusion. The substrate tolerance of the CrpE polypeptide was investigated with a series of structurally related cryptophycin analogues generated by chemoenzymatic synthesis. The enzyme specifically installed a beta-epoxide between C2' and C3' of cyclic cryptophycin analogues. The kcat/Km values of the enzyme were determined to provide further insights into the P450 epoxidase catalytic efficiency affected by substrate structural variation. Finally, binding analysis revealed cooperativity of MBP-CrpE toward natural and unnatural desepoxy cryptophycin substrates.
authors: Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors: Yousong
authors: Y
authors: Ding
authors: Wolfgang H
authors: WH
authors: Seufert
authors: Zachary Q
authors: ZQ
authors: Beck
authors: David H
authors: DH
authors: Sherman
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism; D047630:Depsipeptides / Q000494:pharmacology*; D010088:Oxidoreductases / Q000032:analysis / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D000903:Antibiotics, Antineoplastic; D047630:Depsipeptides; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C023111:epoxidase