Inactivation of Toluene 2-Monooxygenase in Burkholderia cepacia G4 by Alkynes

Abstract

ABSTRACT High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C 5 to C 10 ) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o -cresol-dependent O 2 uptake activities, while acetate- and 3-methylcatechol-dependent O 2 uptake activities were unaffected. Toluene-dependent O 2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min −1 to 2.45 min −1 . Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H 2 O 2 , was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne—ethylene and propylene—were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent K s and V max values of 39.7 μM and 112.3 nmol min −1 mg of protein −1 for ethylene and 32.3 μM and 89.2 nmol min −1 mg of protein −1 for propylene.