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KMID : 0545120050150030652
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Journal of Microbiology and Biotechnology
2005 Volume.15 No. 3 p.652 ~ p.655
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Cloning and Expression of Isocitrate Lyase, a Key Enzyme of the Glyoxylate Cycle, of Candida albicans for Development of Antifungal Drugs
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Shin DS
Kim SH/Yang HC/Oh KB
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Abstract
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This paper describes the development of an enzymatic assay system for the identification of inhibitors of isocitrate lyase (ICL), one of the key enzymes of the glyoxylate cycle that is considered as a new target for antifungal drugs. A 1.6 kb DNA fragment encoding the isocitrate lyase from Candida albicans ATCC10231 was amplified by PCR, cloned into a vector providing His-Patch-thioredoxin-tag at the N-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The molecular mass of the purified ICL was approximately 62 kDa, as determined by SDS-PAGE, and the enzyme activity was directly proportional to incubation time and enzyme concentration. The effects of itaconate-related compounds on ICL activity were also investigated. Among them, itaconic acid, 3-nitropropionate, and oxalate had strong inhibitory activities with IC50 values of 5.8, 5.4, and 8.6 μg/ml, respectively. These inhibitors also exhibited antifungal activity on YPD agar media containing acetate as a sole carbon source, albeit at high concentration. The results indicate that the C. albicans ICL may be a regulatory enzyme playing a crucial role in fungal growth and is a prime target for antifungal agents.
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