Williams N K, Simpson R J, Moritz R L, Peide Y, Crofts L, Minasian E, Leach S J, Wake R G, Christopherson R I
Department of Biochemistry, University of Sydney, New South Wales, Australia.
Gene. 1990 Oct 15;94(2):283-8. doi: 10.1016/0378-1119(90)90399-c.
Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the cDNA from the plasmid pCAD142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the 3'-end of the cDNA. The DHOase and ATCase domains have been purified from an elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Leu followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular DHOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37,751) is separated from the C-terminal ATCase domain (Mr 34,323) by a bridging sequence (Mr 12,532) consisting of multiple beta-turns.
哺乳动物二氢乳清酸酶（DHOase，EC 3.5.2.3）是三功能蛋白二氢乳清酸合成酶的一部分，该酶催化嘧啶从头生物合成的前三个反应。我们从质粒pCAD142中亚克隆了部分cDNA，并获得了一个核苷酸序列，该序列从cDNA 3'端编码天冬氨酸转氨甲酰酶（ATCase）结构域的序列开始，在5'方向上延伸2.1 kb。已从三功能蛋白的弹性蛋白酶消化物中纯化出DHOase和ATCase结构域，并从其N端进行氨基酸（aa）测序。已获得DHOase结构域N端24个aa的序列，并与cDNA序列进行了比对。DHOase结构域的C端残基已被鉴定为Leu，其后是Val，结合该结构域CNBr片段的部分序列，确定了通过蛋白水解释放的活性球状DHOase结构域的编码序列。从推导的aa序列预测蛋白质二级结构表明，DHOase结构域（Mr 37,751）通过由多个β-转角组成的桥接序列（Mr 12,532）与C端ATCase结构域（Mr 34,323）分开。
