Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis
Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis
复制标题
DOI：
10.1042/bj20100597
复制
发表时间：
2010-11-01
影响因子：
4.1
通讯作者：
Peters, Reuben J.
中科院分区：
文献类型：
--
作者：
Morrone, Dana;Chen, Xiaoming;Peters, Reuben J.
KO （kaurene oxidase）是一种多功能细胞色素P450，在赤霉素植物激素生物合成过程中催化三次连续氧化，将对-kaurene烯烃中间体的C4 α甲基转化为对-kaurene -19-oic酸的羧酸部分。为了研究KO的未知催化机制和特性，我们从拟南芥（AtKO）中设计了相应的CYP701A3，用于在大肠杆菌中进行功能重组表达。利用重组AtKO （rAtKO）进行了O-18(2)标记研究，研究了对-kaurene及其中间体对-kaurenol和对-kaurenal的多功能反应序列，揭示了三个羟基化反应的催化作用，这些反应在初始羟基化后的某个阶段需要进一步脱水。然后必须进一步羟基化成宝石二醇中间体，动力学分析表明，这些中间体在反应过程中都保留在活性位点，而第一次羟基化反应是限速的。此外，对其他底物的研究表明，在C4a的末端，对映烯的环结构不同甲基仅被rAtKO羟基化，这表明kaurane精确的四环结构对于KO的多功能活性至关重要，因此本研究的结果明确了KO的反应顺序和酶促机制，以及催化多重反应序列的关键底物特征
KO (kaurene oxidase) is a multifunctional cytochrome P450 catalysing three sequential oxidations in gibberellin phytohormone biosynthesis These serve to transform the C4 alpha methyl of the ent-kaurene olefin intermediate Into the carboxylic acid moiety of ent-kauren-19-oic acid To investigate the unknown catalytic mechanism and properties of KO we have engineered the corresponding CYP701A3 from Arabidopsis thaliana (AtKO) for functional recombinant expression in Escherichia coli, involving use of a fully codon-optimized construct, along with additional N-terminal deletion and modification This recombinant AtKO (rAtKO) was used to carry out O-18(2) labelling studies with ent-kaurene, and the intermediates ent-kaurenol and ent-kaurenal, to investigate the multifunctional reaction sequence, revealing catalysis of three hydroxylation reactions which further requires dehydration at some stage Accordingly, following initial hydroxylation, ent-kaurenol must then be further hydroxylated to a gem diol intermediate, and our data indicate that the subsequent reactions proceed via dehydration of the gem-diol to ent-kaurenal followed by an additional hydroxylation to directly form ent-kaurenoic acid Kinetic analysis indicates that these intermediates are all retained in the active site during the course of the reaction series with the first hydroxylation being rate-limiting In addition investigation of alternative substrates demonstrated that ent-beyerene which differs in ring structure distal to the C4a methyl is only hydroxylated by rAtKO, indicating the importance of the exact tetracyclic ring structure of kaurane for multifunctional KO activity Thus the results of the present study clarify the reaction sequence and enzymatic mechanism of KO, as well as substrate features critical for the catalysed multiple reaction sequence