Ding Yousong, Seufert Wolfgang H, Beck Zachary Q, Sherman David H
Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Am Chem Soc. 2008 Apr 23;130(16):5492-8. doi: 10.1021/ja710520q. Epub 2008 Mar 26.
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.
隐藻素是从 Nostoc sp. ATCC 53789 和 Nostoc sp. GSV 224 中分离出的强效抗癌剂。最有效的天然隐藻素类似物在分子的 C2'-C3' 位置保留一个β-环氧化物。最近从隐藻素基因簇中鉴定出的由 crpE 编码的一种 P450 环氧化酶，能够以区域和立体特异性方式将这个关键官能团引入隐藻素-4（Cr-4）中，生成隐藻素-2（Cr-2）。在此，我们报道了使用工程化麦芽糖结合蛋白（MBP）-CrpE 融合蛋白对 CrpE 环氧化酶进行的详细表征。利用化学酶促合成产生的一系列结构相关的隐藻素类似物研究了 CrpE 多肽的底物耐受性。该酶能特异性地在环状隐藻素类似物的 C2' 和 C3' 之间引入一个β-环氧化物。测定了该酶的 kcat/Km 值，以进一步深入了解受底物结构变化影响的 P450 环氧化酶的催化效率。最后，结合分析揭示了 MBP-CrpE 对天然和非天然去环氧隐藻素底物的协同作用。
