Dal Nogare A R, Dan N, Lehrman M A
Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical Center At Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9041, USA.
Glycobiology. 1998 Jun;8(6):625-32. doi: 10.1093/glycob/8.6.625.
The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B-D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP-GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.
真核生物和原核生物的UDP-GlcNAc/MurNAc家族酶以UDP-GlcNAc或UDP-MurNAc-五肽作为供体，以多萜醇-P或聚异戊二烯醇-P作为受体，并生成糖-P-P-聚异戊二烯醇。在这个家族中已鉴定出一系列六个保守序列，命名为A至F，长度从5到13个氨基酸残基不等。为了确定这些保守序列是否是酶功能所必需的，对仓鼠UDP-GlcNAc:多萜醇-P GlcNAc-1-P转移酶（GPT）进行了各种突变检测。通过打乱每个序列内的残基产生的序列B-F的随机突变表明，每个序列在GPT中都很重要。虽然E和F的随机突变似乎阻止了GPT的稳定表达，但B-D的随机突变产生了可以稳定表达并结合衣霉素的GPT突变体，但缺乏酶活性。此外，C和D的随机突变体有一个意想不到的分选缺陷。用来自枯草芽孢杆菌mraY或大肠杆菌rfe基因的原核对应序列替换序列B-F也影响了GPT，要么阻止突变蛋白的表达（B、F），要么抑制其酶活性（C-E）。对于C-E的替换，未检测到对完全不饱和的天然细菌受体多萜醇-P的受体活性的获得。这些研究表明，UDP-GlcNAc/MurNAc家族的保守序列很重要，并且真核生物和原核生物的对应序列不能自由互换。由于几个突变体能够有效表达并结合衣霉素，但缺乏酶活性，这些数据与这些序列在产物形成中具有直接作用是一致的。
