Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases
Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases
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DOI：
10.1074/jbc.m707411200
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发表时间：
2008-02-15
影响因子：
4.8
通讯作者：
Schofield, Christopher J.
中科院分区：
文献类型：
--
作者：
Flashman, Emily;Bagg, Eleanor A. L.;Schofield, Christopher J.
缺氧诱导因子（HIF）α亚基N-和C-末端氧依赖性降解结构域（NODD和CODD）中两个保守脯氨酰残基的羟基化通过泛素-蛋白酶体途径发出其降解信号。在人类细胞中，属于Fe（II）和2-酮戊二酸（2 OG）依赖性加氧酶家族的三种脯氨酰羟化酶（PHDs 1-3）催化脯氨酰羟化，对CODD和NODD具有不同的选择性。根据PHD 2的晶体结构对PHDs的催化结构域进行的序列分析以及其他2 OG加氧酶的结果表明，C末端区域或连接两条β链（人PHD 2中的β 2和β 3）的环在确定底物选择性方面很重要。对PHD 2的突变分析显示，β 2 β 3环是赋予CODD相对于NODD肽的选择性的主要决定因素。其中PHD 2的β 2 β 3环被PHD 3的β 2 β 3环替换的嵌合PHD 3显示出对CODD几乎完全的选择性（在竞争实验中），如对野生型PHD 3所观察到的。观察到CODD比野生型PHD 2催化结构域更紧密地结合该嵌合蛋白。
Hydroxylation of two conserved prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the alpha-subunit of hypoxia-inducible factor (HIF) signals for its degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1-3) belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two beta-strands (beta 2 and beta 3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the beta 2 beta 3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the beta 2 beta 3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD(in competition experiments), as observed for wild-type PHD3. CODD was observed to bind much more tightly to this chimeric protein than the wild type PHD2 catalytic domain.