Simmer J P, Kelly R E, Rinker A G, Zimmermann B H, Scully J L, Kim H, Evans D R
Department of Biochemistry, Wayne State University, Detroit, MI 48201.
Proc Natl Acad Sci U S A. 1990 Jan;87(1):174-8. doi: 10.1073/pnas.87.1.174.
Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA. The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetase-DHO-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus.
哺乳动物二氢乳清酸酶（S - 二氢乳清酸酰胺水解酶，EC 3.5.2.3）是一种名为CAD的大型多功能蛋白的一部分，该蛋白还具有氨甲酰磷酸合成酶[二氧化碳：L - 谷氨酰胺酰胺连接酶（ADP形成，氨基甲酸酯磷酸化），EC 6.3.5.5]和天冬氨酸转氨甲酰酶（氨甲酰磷酸：L - 天冬氨酸氨甲酰转移酶，EC 2.1.3.2）的活性。我们对叙利亚仓鼠CAD cDNA的选定限制性片段进行了测序。推导的氨基酸序列与胰蛋白酶肽的序列以及通过对CAD进行可控蛋白水解分离出的二氢乳清酸酶结构域的氨基酸组成一致。用含有位于天冬氨酸转氨甲酰酶编码区5'端的cDNA片段的重组质粒转化的大肠杆菌表达了一种能被二氢乳清酸酶结构域特异性抗体识别的多肽。因此，该多肽内各结构域的顺序为NH2 - 氨甲酰磷酸合成酶 - 二氢乳清酸酶 - 天冬氨酸转氨甲酰酶 - COOH。由334个残基组成的二氢乳清酸酶结构域的分子量为36,733，pI为6.1。在胰蛋白酶消化后分离出的具有二氢乳清酸酶活性的CAD片段在该核心结构域的NH2端（18个残基）和COOH端（47 - 65个残基）都有延伸。五个保守组氨酸中的三个位于可能参与锌结合的短的、高度保守的区域内。系统发育分析将单功能和融合的二氢乳清酸酶分别聚类。虽然这些家族可能是通过趋同进化产生的，但我们倾向于一种涉及二氢乳清酸酶基因复制并插入到一个祖先双功能基因座的模型。
