Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase
Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase
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DOI：
10.1074/jbc.m110.147934
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发表时间：
2010-09-17
影响因子：
4.8
通讯作者：
Wilson, Mark A.
中科院分区：
文献类型：
--
作者：
Lakshminarasimhan, Mahadevan;Madzelan, Peter;Wilson, Mark A.
异氰化物（以前称为异腈）水合酶（EC 4.2.1.103）是DJ-1超家族的一种酶，其使异氰化物水合以产生相应的N-甲酰胺。为了了解异氰水合酶（ICH）催化的结构基础，我们确定了野生型和几个定点突变体的荧光假单胞菌ICH的晶体结构，分辨率范围从1.0到1.9埃。我们还开发了一种简单的紫外-可见分光光度法测定ICH活性使用2-萘异腈作为底物。ICH含有催化所需的高度保守的半胱氨酸残基（Cys（101）），并与活性位点中的Asp（17）、Thr（102）和有序水分子相互作用。Asp（17）具有与质子化一致的羧酸键长，我们建议它激活有序的水分子以水合有机异腈。与Cys（101）和Asp（17）相反，Thr（102）耐受诱变，并且T102 V突变导致底物抑制酶。尽管ICH与人DJ-1相似（1.6埃C-α均方根偏差），但Cys（101）附近的结构差异不利于该残基的容易氧化，该残基在人DJ-1中具有重要功能，但对ICH活性有害。ICH活性位点区域也表现出令人惊讶的构象可塑性和样品在晶体中的两种不同的构象。ICH代表了DJ-1超家族的一个先前未表征的进化枝，该进化枝具有新的酶活性，表明DJ-1核心折叠可以通过微妙调节保守的反应性半胱氨酸残基的环境来进化多种功能。
Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several site-directed mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 angstrom. We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys(101)) that is required for catalysis and interacts with Asp(17), Thr(102), and an ordered water molecule in the active site. Asp(17) has carboxylic acid bond lengths that are consistent with protonation, and we propose that it activates the ordered water molecule to hydrate organic isocyanides. In contrast to Cys(101) and Asp(17), Thr(102) is tolerant of mutagenesis, and the T102V mutation results in a substrate-inhibited enzyme. Although ICH is similar to human DJ-1 (1.6 angstrom C-alpha root mean square deviation), structural differences in the vicinity of Cys(101) disfavor the facile oxidation of this residue that is functionally important in human DJ-1 but would be detrimental to ICH activity. The ICH active site region also exhibits surprising conformational plasticity and samples two distinct conformations in the crystal. ICH represents a previously uncharacterized clade of the DJ-1 superfamily that possesses a novel enzymatic activity, demonstrating that the DJ-1 core fold can evolve diverse functions by subtle modulation of the environment of a conserved, reactive cysteine residue.