In vitro stabilization of 6-methylsalicylic acid synthetase from Penicillium urticae.
In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetase in vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesis in vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells of Penicillium urticae rapidly increased just before the loss of 6-methylsalicylic acid synthetase content. These in vitro stabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymes in vivo, and into possible ways of extending the life of these catalysts.
Lam
K S
KS
Department of Biological Sciences, University of Calgary, Alta., Canada.
Neway
J O
JO
Gaucher
G M
GM
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001205:Ascorbic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004355:Drug Stability; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008316:Malonyl Coenzyme A / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010365:Patulin / Q000096:biosynthesis; D010407:Penicillium / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D013438:Sulfhydryl Compounds
D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D008316:Malonyl Coenzyme A; D009249:NADP; D010664:Phenylmethylsulfonyl Fluoride; D000105:Acetyl Coenzyme A; D010365:Patulin; D010088:Oxidoreductases; D000217:Acyltransferases; D010447:Peptide Hydrolases; C021587:6-methylsalicylic acid synthetase; D008025:Ligases; D001205:Ascorbic Acid; D004229:Dithiothreitol