Crystal structures of the wild-type, P1A mutant, and inactivated malonate semialdehyde decarboxylase: A structural basis for the decarboxylase and hydratase activities
Crystal structures of the wild-type, P1A mutant, and inactivated malonate semialdehyde decarboxylase: A structural basis for the decarboxylase and hydratase activities
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DOI：
10.1021/bi051383m
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发表时间：
2005-11-15
期刊：
影响因子：
2.9
通讯作者：
Whitman, CP
中科院分区：
文献类型：
--
作者：
Almrud, JJ;Poelarends, GJ;Whitman, CP
丙二酸半醛脱羧酶（MSAD）是一种利用Pro-1和Arg-75将丙二酸半醛转化为乙醛的互变酶超家族成员。Pro-1和Arg-75也与MSAD的水合酶活性有关，其中2-氧-3-戊酸酯被加工成乙酰丙酮酸酯。MSAD的晶体结构（1.8 (A) /圈分辨率），MSAD的PIA突变体（2.7 (A) /圈分辨率），以及被3-氯丙酸（1.6 (A) /圈分辨率）灭活的MSAD， 3-氯丙酸是一种由MSAD水合酶活性激活的机制抑制剂。对比P1A-MSAD和MSAD的结构，发现它们的几何变化不大，说明Pro-1在P1A-MSAD中起重要的催化作用，而不是关键的结构作用。野生型MSAD和MSAD的结构被3-氧丙酸共价修饰在Pro-1上，3-氧丙酸是MSAD和3-氯丙酸孵化所产生的加合物，这意味着Asp-37是激活水分子攻击3-氯丙酸C-3的残基，从而引发水的Michael加成。Arg-73和Arg-75与加合物C-1羧酸基的相互作用表明，这些残基使α， β -不饱和酸极化，有利于水的加入。基于这些结构，可以建立3-氯丙酸对MSAD的失活机制以及脱羧酶和水合酶活性的失活机制。结果还提供了额外的证据，支持MSAD和反式-1,3-二氯丙烯降解途径中MSAD之前的一个互变酶超家族成员反式-3-氯丙烯酸脱卤酶的假设，它们来自一个共同的祖先，但保留了共轭加水的关键元件。
Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 is a tautomerase superfamily member that converts malonate semialdehyde to acetaldehyde by a mechanism utilizing Pro-1 and Arg-75. Pro-1 and Arg-75 have also been implicated in the hydratase activity of MSAD in which 2-oxo-3-pentynoate is processed to acetopyruvate. Crystal structures of MSAD (1.8 (A) over circle resolution), the PIA mutant of MSAD (2.7 (A) over circle resolution), and MSAD inactivated by 3-chloropropiolate (1.6 (A) over circle resolution), a mechanism-based inhibitor activated by the hydratase activity of MSAD, have been determined. A comparison of the P1A-MSAD and MSAD structures reveals little geometric alteration, indicating that Pro-1 plays an important catalytic role but not a critical structural role. The structures of wild-type MSAD and MSAD covalently modified at Pro-1 by 3-oxopropanoate, the adduct resulting from the incubation of MSAD and 3-chloropropiolate, implicate Asp-37 as the residue that activates a water molecule for attack at C-3 of 3-chloropropiolate to initiate a Michael addition of water. The interactions of Arg-73 and Arg-75 with the C-1 carboxylate group of the adduct suggest these residues polarize the alpha,beta-unsaturated acid and facilitate the addition of water. On the basis of these structures, a mechanism for the inactivation of MSAD by 3-chloropropiolate can be formulated along with mechanisms for the decarboxylase and hydratase activities. The results also provide additional evidence supporting the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, a tautomerase superfamily member preceding MSAD in the trans-1,3-dichloropropene degradation pathway, diverged from a common ancestor but retained the key elements for the conjugate addition of water.