Analysis of the cryptophycin P450 epoxidase reveals substrate tolerance and cooperativity
Analysis of the cryptophycin P450 epoxidase reveals substrate tolerance and cooperativity
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DOI：
10.1021/ja710520q
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发表时间：
2008-04-23
影响因子：
15
通讯作者：
Sherman, David H.
中科院分区：
文献类型：
--
作者：
Ding, Yousong;Seufert, Wolfgang H.;Sherman, David H.
念珠藻素是分离自念珠藻属ATCC 53789和念珠藻属GSV 224的有效抗癌剂。最有效的天然念珠藻素类似物在分子的C2 '-C3'位置保留β-环氧化物。最近从念珠藻素基因簇中鉴定出的crpE编码的P450环氧酶被证明以区域和立体特异性的方式将该关键官能团安装到念珠藻素-4（Cr-4）中以产生念珠藻素-2（Cr-2）。在这里，我们报告了使用工程麦芽糖结合蛋白（MBP）-CrpE融合的CrpE环氧酶的详细表征。CrpE多肽的底物耐受性进行了研究与化学酶合成产生的一系列结构相关的念珠藻素类似物。该酶特异性地在环状念珠藻素类似物的C2'和CT之间安装O-环氧化物。确定酶的k（cat）/K-m值，以进一步了解底物结构变化对P450环氧酶催化效率的影响。最后，结合分析揭示了MBP-CrpE对天然和非天然脱环氧念珠藻素底物的协同性。
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 crpE 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 O-epoxide between C2' and CT of cyclic cryptophycin analogues. The k(cat)/K-m 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.