Williams N K, Peide Y, Seymour K K, Ralston G B, Christopherson R I
Department of Biochemistry, University of Sydney, NSW, Australia.
Protein Eng. 1993 Apr;6(3):333-40. doi: 10.1093/protein/6.3.333.
Dihydroorotase is the central domain of trifunctional L-dihydroorotate synthetase which also contains carbamyl phosphate synthetase at the N-terminus and aspartate transcarbamylase at the C-terminus. The cDNA, corresponding to the active dihydroorotase domain as isolated after digestion of dihydroorotate synthetase with elastase, has been sub-cloned into the expression vector pCW12 which was then used to transform Escherichia coli S phi 1263 pyrC- lacking dihydroorotase activity. However, induction of this recombinant strain with IPTG produced large amounts of the dihydroorotase domain which were completely inactive. A number of cDNAs were expressed which were longer on the C-terminal side; all cDNAs expressed active dihydroorotase domain down to a minimal extension of 12 amino acids (-Val-Pro-Pro-Gly-Tyr-Gly-Gln-Asp-Val-Arg-Lys-Trp) into the bridge region between the dihydroorotase and aspartate transcarbamylase domains. Part of this dodecapeptide may form an amphipathic helix which in some way constrains the isolated, recombinant dihydroorotase domain to an active conformation. The recombinant hamster dihydroorotase purified from a cell-free extract of E. coli in four steps has a turnover number of 297 mol/min/(mol domain) for the conversion of L-dihydroorotate back to N-carbamyl-L-aspartate with Ks = 8.7 +/- 1.5 microM for L-dihydroorotate, a subunit molecular weight of 39,008 determined from the sequence and 37,900 +/- 400 when subjected to SDS-PAGE, and an isoelectric point of 5.7. Ultracentrifugal analysis of the recombinant domain showed a single species of s20,w = 4.1 S and a single molecular species of M(r) = 76,000 corresponding to a dimer.
二氢乳清酸酶是三功能L-二氢乳清酸合成酶的中心结构域，该合成酶在N端还含有氨甲酰磷酸合成酶，在C端含有天冬氨酸转氨甲酰酶。用弹性蛋白酶消化二氢乳清酸合成酶后分离得到的与活性二氢乳清酸酶结构域对应的cDNA，已亚克隆到表达载体pCW12中，然后用于转化缺乏二氢乳清酸酶活性的大肠杆菌S phi 1263 pyrC-。然而，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导该重组菌株产生了大量完全无活性的二氢乳清酸酶结构域。表达了一些在C端更长的cDNA；所有表达的cDNA都具有活性二氢乳清酸酶结构域，其在二氢乳清酸酶和天冬氨酸转氨甲酰酶结构域之间的桥区域中最小延伸12个氨基酸（-缬氨酸-脯氨酸-脯氨酸-甘氨酸-酪氨酸-甘氨酸-谷氨酰胺-天冬氨酸-缬氨酸-精氨酸-赖氨酸-色氨酸）时仍保持活性。这个十二肽的一部分可能形成一个两亲性螺旋，它以某种方式将分离的重组二氢乳清酸酶结构域限制为活性构象。从大肠杆菌的无细胞提取物中通过四个步骤纯化的重组仓鼠二氢乳清酸酶，将L-二氢乳清酸转化回N-氨甲酰-L-天冬氨酸的周转数为297 mol/min/（mol结构域），对L-二氢乳清酸的Ks = 8.7±1.5 microM，根据序列确定的亚基分子量为39,008，在进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）时为37,900±400，等电点为5.7。对重组结构域的超速离心分析显示，单一物种的沉降系数s20,w = 4.1 S，单一分子物种的相对分子质量M(r) = 76,000，对应于一个二聚体。
