Initial reactions in anaerobic oxidation of m-xylene by the denitrifying bacterium Azoarcus sp. strain T.
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.
Krieger
C J
CJ
Environmental Engineering, Department of Civil Engineering, Stanford University, Stanford, California 94305-4020, USA.
Beller
H R
HR
Reinhard
M
M
Spormann
A M
AM
D000693:Anaerobiosis; D020627:Azoarcus / Q000145:classification / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism; D001673:Biodegradation, Environmental; D002463:Cell Membrane Permeability; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D010802:Phylogeny; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014050:Toluene / Q000378:metabolism; D014992:Xylenes / Q000378:metabolism*
D001565:Benzoates; D009566:Nitrates; D009573:Nitrites; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D014992:Xylenes; C009503:3-toluic acid; D014050:Toluene; C031286:4-xylene; C031285:3-xylene; C026114:2-xylene