Initial Reactions in Anaerobic Oxidation of m -Xylene by the Denitrifying Bacterium Azoarcus sp. Strain T

Abstract

ABSTRACT The initial enzymatic steps in anaerobic m -xylene oxidation were studied in Azoarcus sp. strain T, a denitrifying bacterium capable of mineralizing m -xylene via 3-methylbenzoate. Permeabilized cells of m -xylene-grown Azoarcus sp. strain T catalyzed the addition of m -xylene to fumarate to form (3-methylbenzyl)succinate. In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to E -(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate. Kinetic studies conducted with permeabilized cells and whole-cell suspensions of m -xylene-grown Azoarcus sp. strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo m -xylene consumption. Based on these findings, we propose that Azoarcus sp. strain T anaerobically oxidizes m -xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and E -(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA. A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond.