Weinberg R A, McWherter C A, Freeman S K, Wood D C, Gordon J I, Lee S C
Department of Research and Development, G.D. Searle and Company, St. Louis, Missouri 63198, USA.
Mol Microbiol. 1995 Apr;16(2):241-50. doi: 10.1111/j.1365-2958.1995.tb02296.x.
MyristoylCoA:protein N-myristoyltransferase (Nmt) catalyses the co-translational, covalent attachment of myristate (C14:0) to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction. The NMT1 gene is essential for vegetative growth of Saccharomyces cerevisiae. Studies were carried out to determine if Nmt is also essential for vegetative growth of the pathogenic fungus Candida albicans. A strain of C. albicans was constructed in which one copy of NMT was partially deleted and disrupted. A Gly-447-->Asp mutation was introduced into the second NMT allele. This mutation produced marked reductions in catalytic efficiency at 24 and 37 degrees C, as judged by in vitro kinetic studies of the wild-type and mutant enzymes which had been expressed in, and purified from, Escherichia coli. The growth characteristics of isogenic NMT/NMT, NMT/delta nmt, and nmt delta/nmtG447D C. albicans strains were assessed under a variety of conditions. Only the nmt delta/nmtG447D strain required myristate for growth. This was true at both 24 and 37 degrees C. Palmitate could not substitute for myristate. Incubation of nmt delta/nmtG447D cells at 37 degrees C in the absence of myristate resulted in cell death as observed by the inability to form colonies on media supplemented with 500 microM myristate. Studies in an immunosuppressed-mouse model of C. albicans infection revealed that the NMT/delta nmt strain produced 100% lethality within 7 d after intravenous administration while the isogenic nmt delta/nmtG447G strain produced no deaths even after 21 d. These observations establish that Nmt is essential for vegetative growth of C. albicans and suggest that inhibitors of this acyltransferase may be therapeutically useful fungicidal agents.
肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶（Nmt）催化肉豆蔻酸（C14:0）与许多参与细胞生长和信号转导的真核蛋白质的氨基末端甘氨酸残基进行共翻译的共价连接。NMT1基因对于酿酒酵母的营养生长至关重要。开展了研究以确定Nmt对于致病性真菌白色念珠菌的营养生长是否也必不可少。构建了一株白色念珠菌，其中一个NMT拷贝被部分缺失和破坏。在第二个NMT等位基因中引入了Gly-447→Asp突变。通过对在大肠杆菌中表达并纯化的野生型和突变型酶进行体外动力学研究判断，该突变在24℃和37℃时使催化效率显著降低。在多种条件下评估了同基因的NMT/NMT、NMT/δnmt和nmtδ/nmtG447D白色念珠菌菌株的生长特性。只有nmtδ/nmtG447D菌株生长需要肉豆蔻酸。在24℃和37℃时均是如此。棕榈酸不能替代肉豆蔻酸。在没有肉豆蔻酸的情况下于37℃孵育nmtδ/nmtG447D细胞导致细胞死亡，这可通过在补充有500μM肉豆蔻酸的培养基上无法形成菌落观察到。在白色念珠菌感染的免疫抑制小鼠模型中的研究表明，NMT/δnmt菌株在静脉注射后7天内产生100%的致死率，而同基因的nmtδ/nmtG447G菌株即使在21天后也未导致死亡。这些观察结果表明Nmt对于白色念珠菌的营养生长至关重要，并提示这种酰基转移酶的抑制剂可能是具有治疗用途的杀真菌剂。
