Xu Y, Mitra B
Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
Biochemistry. 1999 Sep 21;38(38):12367-76. doi: 10.1021/bi990996u.
(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, is a membrane-associated protein, in contrast to the more well-characterized members of this protein family including glycolate oxidase (GOX) from spinach. In a previous study [Mitra, B., et al. (1993) Biochemistry 32, 12959-12967], the membrane association of MDH was correlated to a 53 amino acid segment in the interior of the primary sequence by construction of a chimeric enzyme, MDH-GOX1, in which the membrane-binding segment in MDH was deleted and replaced with the corresponding 34 amino acid segment from the soluble GOX. Though MDH-GOX1 was soluble, it was an inefficient, nonspecific enzyme that involved a different transition state for the catalyzed reaction from that of the wild-type MDH. In the present study, it is shown that the membrane-binding segment in MDH is somewhat shorter, approximately 39 residues long. Partial or total deletion of this segment disrupts membrane localization of MDH. This segment is not important for substrate oxidation activity. A new chimera, MDH-GOX2, was created by replacing this shorter membrane-binding segment from MDH with the corresponding 20 amino acid segment from GOX. The soluble MDH-GOX2 is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toward oxygen. Therefore, it should prove to be a highly useful model for structural studies of MDH.
恶臭假单胞菌的(S)-扁桃酸脱氢酶(MDH)是黄素单核苷酸依赖性α-羟基酸氧化酶/脱氢酶家族的成员,它是一种膜相关蛋白,这与该蛋白家族中其他特征更明确的成员不同,比如菠菜中的乙醇酸氧化酶(GOX)。在之前的一项研究中[米特拉,B.等人(1993年)《生物化学》第32卷,第12959 - 12967页],通过构建嵌合酶MDH - GOX1,将MDH的膜结合片段删除并用可溶性GOX的相应34个氨基酸片段取代,从而将MDH的膜结合与一级序列内部的一个53个氨基酸的片段关联起来。虽然MDH - GOX1是可溶的,但它是一种低效、非特异性的酶,其催化反应的过渡态与野生型MDH不同。在本研究中,结果表明MDH中的膜结合片段稍短,大约有39个残基长。该片段的部分或全部缺失会破坏MDH的膜定位。该片段对底物氧化活性并不重要。通过用GOX的相应20个氨基酸片段替换MDH中这个较短的膜结合片段,创建了一种新的嵌合体MDH - GOX2。可溶性的MDH - GOX2在光谱性质、底物特异性、催化活性、动力学机制以及对氧的反应性方面与野生型膜结合酶非常相似。因此,它应该被证明是用于MDH结构研究的非常有用的模型。