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 (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).
Department of Biology, Washington University, St. Louis, MO 63130, USA.
Ching-Nen
CN
Chen
Larysa
L
Porubleva
Georgia
G
Shearer
Maja
M
Svrakic
Lauren G
LG
Holden
James L
JL
Dover
Mark
M
Johnston
Parag R
PR
Chitnis
Daniel H
DH
Kohl
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000429:Alcohol Oxidoreductases; C031416:D-lactaldehyde dehydrogenase