Schimoler-O'Rourke Rebecca, Renault Samantha, Mo Wenjun, Selitrennikoff Claude P
Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, 4200 E 9th Ave, Denver, CO 80262, USA.
Curr Microbiol. 2003 Jun;46(6):408-12. doi: 10.1007/s00284-002-3884-5.
The essential fungal cell-wall polymer (1,3)beta-glucan is synthesized by the enzyme (1,3)beta-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)beta-glucan synthase. We have purified (1,3)beta-glucan synthase from Neurospora crassa approximately 400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[beta-(32)P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H(+)-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)beta-glucan synthase.
重要的真菌细胞壁聚合物（1,3）-β-葡聚糖由（1,3）-β-葡聚糖合酶合成。这种酶是棘白菌素和杀真菌抗生素类的肺炎球菌素的作用靶点，是一种至少由两种蛋白质Rho1p和Fks1p组成的复合物。在许多真菌中都发现了酵母FKS1基因的同源物，现有证据表明但尚未证明Fks1p是（1,3）-β-葡聚糖合酶的催化亚基。我们已从粗糙脉孢菌中纯化出（1,3）-β-葡聚糖合酶，富集倍数约为400倍，并使用UDP-葡萄糖类似物5-叠氮基-[β-（32）P]-UDP-葡萄糖标记底物结合蛋白。UDP-葡萄糖结合蛋白与底物类似物进行光交联，并通过对胰蛋白酶肽段测序，利用四极杆飞行时间质谱从SDS-PAGE凝胶中鉴定出来。两种质膜蛋白被标记为FKS和H（+）-ATP酶。这些结果表明FKS似乎是（1,3）-β-葡聚糖合酶的底物结合亚基。
