Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeast Saccharomyces cerevisiae

Methylglyoxal is associated with a broad spectrum of biological effects, including cytostatic and cytotoxic activities. It is detoxified by the glyoxylase system or by its reduction to lactaldehyde by methylglyoxal reductase. We show that methylglyoxal reductase (NADPH‐dependent) is encoded by GRE2...

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Published in:	Yeast (Chichester, England) Vol. 20; no. 6; pp. 545 - 554
Main Authors:	Chen, Ching‐Nen, Porubleva, Larysa, Shearer, Georgia, Svrakic, Maja, Holden, Lauren G., Dover, James L., Johnston, Mark, Chitnis, Parag R., Kohl, Daniel H.
Format:	Journal Article
Language:	English
Published:	Chichester, UK John Wiley & Sons, Ltd 30-04-2003
Subjects:	Alcohol Oxidoreductases - chemistry Alcohol Oxidoreductases - genetics Alcohol Oxidoreductases - isolation & purification Alcohol Oxidoreductases - metabolism GRE2 GRE2 gene GST fusions MALDI–TOF mass spectrometry methylglyoxal methylglyoxal reductase Open Reading Frames Saccharomyces cerevisiae Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae - genetics Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization YOL151w
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Summary:	Methylglyoxal is associated with a broad spectrum of biological effects, including cytostatic and cytotoxic activities. It is detoxified by the glyoxylase system or by its reduction to lactaldehyde by methylglyoxal reductase. We show that methylglyoxal reductase (NADPH‐dependent) is encoded by GRE2 (YOL151w). We associated this activity with its gene by partially purifying the enzyme and identifying by MALDI–TOF the proteins in candidate bands on SDS–PAGE gels whose relative intensities correlated with specific activity through three purification steps. The candidate proteins were then purified using a glutathione‐S‐transferase tag that was fused to them, and tested for methylglyoxal reductase activity. The advantage of this approach is that only modest protein purification is required. Our approach should be useful for identifying many of the genes that encode the metabolic pathway enzymes that have not been associated with a gene (about 275 in S. cerevisiae, by our estimate). Copyright © 2003 John Wiley & Sons, Ltd.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0749-503X 1097-0061
DOI:	10.1002/yea.979