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Applied and Environmental Microbiology, February 1999, p. 632-639, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Chris M. Yeager,1 Peter J. Bottomley,1,2 Daniel J. Arp,1,3 and Michael R. Hyman3,*

Molecular and Cellular Biology Program,1 Department of Botany and Plant Pathology,3 and Department of Microbiology and Crop and Soil Sciences,2 Oregon State University, Corvallis, Oregon 97331-2902

Received 13 July 1998/Accepted 2 November 1998

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 (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-grown B. 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 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 Ks and Vmax 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.

* Corresponding author. Present address: Department of Microbiology, North Carolina State University, Box 7615, Raleigh, NC 27695-7615. Phone: (919) 515-7814. Fax: (919) 515-7867. E-mail: mhyman{at}mbio.ncsu.edu.

Applied and Environmental Microbiology, February 1999, p. 632-639, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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