Abstract
The metabolism of coumarin by a strain of Pseudomonas isolated from soil which utilizes coumarin as a sole carbon source was studied. The metabolic pathway was shown to be coumarin_??_dihydrocoumarin_??_melilotic acid_??_2, 3-dihydroxyphenylpropionic acid, based on the results of (1) isolation and identification of metabolic products, (2) survey on the utilization of the postulated intermeidates and (3) examination of enzymatic reaction. An alternative pathway involving o-coumaric acid and 2, 3-dihydroxycinnamic acid as intermediates at the metabolism of coumarin was also discussed.
Coumarin reducing enzyme (dihydrocoumarin: NAD [NADP] oxydo-reductase) which catalizes the reduction of coumarin to dihydrocoumarin was partially purified from the extracts of the above strain of Pseudomonas and some properties of the enzyme were investigated. The optimum pH of the reaction was 5.25. The enzyme is highly specific with respect to coumarin, and Km values for coumarin and NADH were 6.6×10-6M and 4.1×10-5M, respectively. The enzyme activity was extremely sensitive to sulfhydryl reagents particularly to p-chloromercuribenzoate. 2-Mercaptoethanol or dithiothreitol protected the enzyme from inactivation by low temperature storage. The molecular weight of enzyme was estimated to be about 140, 000 by gel permiation chromatographic method. The enzyme showed a substrate inhibition at higher concentrations of coumarin. This inhibition was noncompetitive with respect to NADH. The enzyme was also inhibited by many coumarin analogues. 3-Hydroxycoumarin showed noncompetitive inhibition with both coumarin and NADH. The mechanism of inhibition for the enzyme is discussed. It is concluded that enzyme protein contains zinc atom and that NADH is attached to zinc in the enzyme reaction.
