Molecular cloning, expression in Escherichia coli, and characterization of a novel l-3-hydroxyacyl coenzyme A dehydrogenase from pig liver

Xue-Ying He, Song-Yu Yang
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引用次数: 13

Abstract

A novel l-3-hydroxyacyl-CoA dehydrogenase from pig liver has been cloned, expressed, purified, and characterized. This enzyme is a homodimer with a molecular mass of 65.6 kDa, and is distinguished from the dehydrogenase of pig heart by its structural features and catalytic properties. Its subunit, consisting of 302 amino acid residues, has two additional residues in a key region of the active center while it lacks a sequence of seven residues in the NAD+-binding domain, when compared with the subunit of pig heart enzyme. In addition, there are substitutions of four single residues. The catalytic efficiency of pig liver dehydrogenase was significantly greater than that of the heart enzyme for short-chain substrate, but its catalytic rates declined with an increase in substrate chain-lengths. The distinction between pig liver and heart dehydrogenases cannot be attributed to a species difference, and thus it is concluded that there exist different isoforms of monofunctional l-3-hydroxyacyl-CoA dehydrogenases in pig. High level expression of mitochondrial l-3-hydroxyacyl-CoA dehydrogenase in Escherichia coli has provided a very convenient way to purify this important β-oxidation enzyme. There is substantial homology between pig liver dehydrogenase and various multifunctional β-oxidation enzymes in the active center of these enzymes; a consensus sequence, HX3PX1–3MXLXE, is proposed as the signature sequence of l-3-hydroxyacyl-CoA dehydrogenases.

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猪肝l-3-羟基酰基辅酶a脱氢酶的克隆、在大肠杆菌中的表达及特性研究
从猪肝中克隆、表达、纯化并鉴定了一种新的l-3-羟基酰基辅酶A脱氢酶。该酶为同二聚体,分子量为65.6 kDa,其结构特征和催化性能与猪心脏脱氢酶相区别。与猪心脏酶亚基相比,其亚基由302个氨基酸残基组成,在活性中心的关键区域多了2个残基,而在NAD+结合区域缺少7个残基序列。此外,还有四个单残基的取代。猪肝脱氢酶对短链底物的催化效率显著大于心脏酶,但随着底物链长度的增加,其催化效率下降。猪肝脏脱氢酶和猪心脏脱氢酶的差异不能归因于物种差异,因此可以得出结论,猪体内存在不同的单功能l-3-羟基酰基辅酶a脱氢酶同工型。线粒体l-3-羟基酰基辅酶a脱氢酶在大肠杆菌中的高水平表达为纯化这种重要的β-氧化酶提供了一种非常方便的方法。猪肝脱氢酶活性中心与多种多功能β-氧化酶具有较强的同源性;一致的序列HX3PX1-3MXLXE被认为是l-3-羟基酰基辅酶a脱氢酶的特征序列。
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