Purification and properties of L-mandelate-4-hydroxylase from Pseudomonas convexa

Abstract

An inducible L-mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of l-mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe²⁺, and O₂ for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with L-mandelate, NADPH, and ferrous sulfate and K_m values for these substrates were found to be 1 × 10^-4, 1.9 × 10^-4, and 4.7 × 10^-5M, respectively. The enzyme is very specific for l-mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.