Rowland P, Björnberg O, Nielsen F S, Jensen K F, Larsen S
Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Denmark.
Protein Sci. 1998 Jun;7(6):1269-79. doi: 10.1002/pro.5560070601.
Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.
二氢乳清酸脱氢酶(DHODs)催化(S)-二氢乳清酸氧化为乳清酸,这是嘧啶核苷酸从头生物合成中的第四步也是唯一的氧化还原反应。本文描述了乳酸乳球菌二氢乳清酸脱氢酶A(DHODA)与酶反应产物乳清酸形成的复合物的晶体结构。已将该复合物分辨率为2.0 Å的结构与天然酶的结构进行了比较。已知DHODA的活性位点包含一个位于高度保守且灵活的环下方的充满水的腔。在复合物中,乳清酸取代了活性位点中的水分子,并堆积在DHODA黄素异咯嗪环上方,仅导致周围蛋白质残基发生微小移动。乳清酸完全埋藏在蛋白质表面之下,并且乳清酸的结合导致活性位点环的流动性显著降低。乳清酸由四个保守的天冬酰胺侧链(天冬酰胺67、天冬酰胺127、天冬酰胺132和天冬酰胺193)、赖氨酸43和丝氨酸194的侧链以及甲硫氨酸69、甘氨酸70和亮氨酸71的主链NH基团结合。其中,赖氨酸43侧链与黄素异咯嗪环和乳清酸的羧基均形成氢键。以乳清酸复合物为分子建模基础,考虑了与结合的二氢乳清酸的潜在相互作用。讨论了半胱氨酸130作为活性位点碱基的作用,并综述了不同DHOD家族活性位点残基的序列保守性,以及这些家族在底物结合和催化机制上的差异。