Garrad R C, Bhattacharjee J K
Department of Microbiology, Miami University, Oxford, Ohio 45056.
J Bacteriol. 1992 Nov;174(22):7379-84. doi: 10.1128/jb.174.22.7379-7384.1992.
The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. Until now, this unique metabolic pathway has never been investigated in the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. Five of the eight enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase) of the alpha-aminoadipate pathway and glucose-6-phosphate dehydrogenase, a glycolytic enzyme used as a control, were demonstrated in wild-type cells of these organisms. All enzymes were present in Saccharomyces cerevisiae and the pathogenic organisms except C. neoformans 32608 serotype C, which exhibited no saccharopine reductase activity. The levels of enzyme activity varied considerably from strain to strain. Variation among organisms was also observed for the control enzyme. Among the pathogens, C. albicans exhibited much higher homocitrate synthase, homoisocitrate dehydrogenase, and alpha-aminoadipate reductase activities. Seven lysine auxotrophs of C. albicans and one of Candida tropicalis were characterized biochemically to determine the biochemical blocks and gene-enzyme relationships. Growth responses to alpha-aminoadipate- and lysine-supplemented media, accumulation of alpha-aminoadipate semialdehyde, and the lack of enzyme activity revealed that five of the mutants (WA104, WA153, WC7-1-3, WD1-31-2, and A5155) were blocked at the alpha-aminoadipate reductase step, two (STN57 and WD1-3-6) were blocked at the saccharopine dehydrogenase step, and the C. tropicalis mutant (X-16) was blocked at the saccharopine reductase step. The cloned LYS1 gene of C. albicans in the recombinant plasmid YpB1078 complemented saccharopine dehydrogenase (lys1) mutants of S. cerevisiae and C. albicans. The Lys1+ transformed strains exhibited significant saccharopine dehydrogenase activity in comparison with untransformed mutants. The cloned LYS1 gene has been localized on a 1.8-kb HindIII DNA insert of the recombinant plasmid YpB1041RG1. These results established the gene-enzyme relationship in the second half of the alpha-aminoadipate pathway. The presence of this unique pathway in the pathogenic fungi could be useful for their rapid detection and control.
赖氨酸生物合成的α-氨基己二酸途径仅存在于真菌和眼虫中。到目前为止，这一独特的代谢途径从未在机会性真菌病原体白色念珠菌、新型隐球菌和烟曲霉中进行过研究。在这些生物体的野生型细胞中证实了α-氨基己二酸途径的八种酶中的五种（高柠檬酸合酶、高异柠檬酸脱氢酶、α-氨基己二酸还原酶、酵母氨酸还原酶和酵母氨酸脱氢酶）以及作为对照的糖酵解酶葡萄糖-6-磷酸脱氢酶。除了新型隐球菌32608 C血清型没有酵母氨酸还原酶活性外，所有酶都存在于酿酒酵母和致病生物体中。酶活性水平因菌株而异。对照酶在生物体之间也观察到差异。在病原体中，白色念珠菌表现出更高的高柠檬酸合酶、高异柠檬酸脱氢酶和α-氨基己二酸还原酶活性。对白色念珠菌的七个赖氨酸营养缺陷型和热带念珠菌的一个营养缺陷型进行了生化表征，以确定生化阻断和基因-酶关系。对添加α-氨基己二酸和赖氨酸的培养基的生长反应、α-氨基己二酸半醛的积累以及酶活性的缺乏表明，五个突变体（WA104、WA153、WC7-1-3、WD1-31-2和A(5155)）在α-氨基己二酸还原酶步骤被阻断，两个（STN57和WD1-3-6）在酵母氨酸脱氢酶步骤被阻断，热带念珠菌突变体（X-16）在酵母氨酸还原酶步骤被阻断。重组质粒YpB1078中白色念珠菌的克隆LYS1基因补充了酿酒酵母和白色念珠菌的酵母氨酸脱氢酶（lys1）突变体。与未转化的突变体相比，Lys1+转化菌株表现出显著的酵母氨酸脱氢酶活性。克隆的LYS1基因已定位在重组质粒YpB1041RG1的1.8 kb HindIII DNA插入片段上。这些结果确立了α-氨基己二酸途径后半部分的基因-酶关系。致病真菌中这种独特途径的存在可能有助于它们的快速检测和控制。
