Conversion of p-coumarate into caffeate by Streptomyces nigrifaciens. Purification and properties of the hydroxylating enzyme.
1. An enzyme responsible for the conversion of p-coumarate into caffeate was purified 97-fold from Streptomyces nigrifaciens. The enzyme had a molecular weight of 18000 as determined by Sephadex G-100 gel filtration and was homogeneous on polyacrylamide-gel electrophoresis. 2. The preparation exhibited both p-coumarate hydroxylase and caffeate oxidase activities. 3. Stoicheiometry of the reaction indicated a mono-oxygenase-mediated catalysis consuming 1mol of O(2)/mol of substrate hydroxylated. 4. NADH, NADPH, tetrahydropteroylglutamate or ascorbate act as electron donors for the reaction, ascorbate being inhibitory at higher concentrations. 5. The optimum enzyme activity was at about pH7.7 and 40 degrees C, with an activation energy of 39kJ/mol. 6. Monophenols such as p-hydroxyphenylpropionate, p-hydroxyphenylacetate, l-tyrosine and dl-p-hydroxyphenyl-lactate were also hydroxylated by the preparation, in addition to p-coumarate. 7. The enzyme was a copper protein having 0.38% copper in a bound form. 8. Thiol-group inhibitors did not affect the reaction. 9. The relationship of the enzyme to other hydroxylases is discussed.
D001205:Ascorbic Acid; D002850:Chromatography, Gel; D002934:Cinnamates / Q000096:biosynthesis*; D003300:Copper / Q000032:analysis; D003374:Coumarins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005492:Folic Acid; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008970:Molecular Weight; D009243:NAD; D009249:NADP; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D010636:Phenols / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism
D002934:Cinnamates; D003374:Coumarins; D010636:Phenols; D009243:NAD; D009249:NADP; D003300:Copper; D005492:Folic Acid; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D001205:Ascorbic Acid