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

Abstract

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepaciaG4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) 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-grownB. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected. Toluene-dependent O2 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 H2O2, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase inB. 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 apparentKs 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.

Aerobic biodegradation of N‐nitrosodimethylamine (NDMA) by axenic bacterial strains

“…Unlike the other naphthol-producing incubations, the addition of acetylene gas did not inhibit this bacterium's production of naphthol. The null effect of acetylene on monooxygenase activity on strain G4 has been documented previously (Yeager et al, 1999). Conversely, strain mt-2 tested negative for both NDMA degradation and the naphthol assay, highlighting the unusual nature of the sidechain TMO when compared to the other toluene monooxygenases.…”

“…Functional differences have previously been observed between T2MO and T4MO. For instance, the monooxygenase activity in strain G4 has been shown to be unaffected by exposure to acetylene gas and phenylacetylene, while KR1 and PKO1 experienced near complete inhibition of toluene monooxygenase activity or growth after brief incubations with these respective substrates (Keener et al, 2001;Yeager et al, 1999). Of course, the most obvious difference between these enzymes is that they hydroxylate different regions of the target toluene molecule (Fig.…”

Differential inhibition in vivo of ammonia monooxygenase, soluble methane monooxygenase and membrane‐associated methane monooxygenase by phenylacetylene

“…An additional criterion is that the rate of inactivation of sMMO activity should be proportional to phenylacetylene at low concentrations, but become saturated at high concentrations. As seen in studies of mechanism-based inhibitors of AMO and toluene-2- monooxygenase (Keener et al, 1998;Yeager et al, 1999), inhibition of sMMO activity increased proportionally with phenylacetylene, but saturation of the observed inactivation rate was not found over the tested concentration range. It is unclear why saturation was not observed, although it may occur at higher concentrations.…”

“…It is unclear why saturation was not observed, although it may occur at higher concentrations. As suggested for the inactivation of toluene-2-monooxygenase by 1-butyne (Yeager et al, 1999), it is possible that the maximal rate of inactivation is faster than the rate at which phenylacetylene binds to sMMO, or that a conventional enzyme±inactivator complex might not be formed before inactivation. Finally, a 1:1 stoichiometry of labelled phenylacetylene to the active site of sMMO must also be demonstrated to verify phenylacetylene as a mechanistic inactivator of sMMO.…”

Adventitious reactions of alkene monooxygenase reveal common reaction pathways and component interactions among bacterial hydrocarbon oxygenases

“…In addition to these oxygenation reactions, several well characterized binuclear iron‐centre monooxygenases have been found to exhibit two adventitious reactions: turnover‐dependent inhibition by alkynes and the so‐called peroxide shunt reaction. Terminal alkynes such as ethyne have been shown to act as suicide substrates of sMMO [19], soluble butane monooxygenase [20], several aromatic monooxygenases [21,22] and the AMO from Xanthobacter sp. Py2 [12,23], presumably by oxygenation to ketenes that then covalently modify and inactivate the enzymes [19].…”