Propionate inactivation of butane monooxygenase activity in ‘Pseudomonas butanovora’: biochemical and physiological implications

Abstract

Butane monooxygenase (BMO) catalyses the oxidation of alkanes to alcohols in the alkane-utilizing bacterium 'Pseudomonas butanovora'. Incubation of alkane-grown 'P. butanovora' with butyrate or propionate led to irreversible time-and O 2 -dependent loss of BMO activity. In contrast, BMO activity was unaffected by incubation with lactate or acetate. Chloramphenicol inhibited the synthesis of new BMO, but did not change the kinetics of propionate-dependent BMO inactivation, suggesting that the propionate effect was not simply due to it acting as a repressor of BMO transcription. BMO was protected from propionate-dependent inactivation by the presence of its natural substrate, butane. Although both the time and O 2 dependency of propionate inactivation of BMO imply that propionate might be a suicide substrate, no evidence was obtained for BMO-dependent propionate consumption, or 14 C labelling of BMO polypeptides by [2-14 C]propionate during inactivation. Propionate-dependent BMO inactivation was also explored in mutant strains of 'P. butanovora' containing single amino acid substitutions in the a-subunit of the BMO hydroxylase. Propionate-dependent BMO inactivation in two mutant strains with amino acid substitutions close to the catalytic site differed from wild-type (one was more sensitive and the other less), providing further evidence that propionate-dependent inactivation involves interaction with the BMO catalytic site. A putative model is presented that might explain propionate-dependent inactivation of BMO when framed within the context of the catalytic cycle of the closely related enzyme, soluble methane monooxygenase.

Degradation of alkanes by bacteria

“…This avoids induction by compounds that are not substrates for BMO. Propionate, the final product of propane oxidation, acts as a potent repressor of BMO operon transcription (Doughty et al ., 2006) and as a direct inhibitor of BMO activity (Doughty et al ., 2007), an effect that persists until propionate catabolism is induced. Propionate catabolism is inactive during growth on butane, but is activated by propionate or upon growth on propane or pentane.…”

The Interaction between Plants and Bacteria in the Remediation of Petroleum Hydrocarbons: An Environmental Perspective

“…For example, expression of BMO in P. butanovora is repressed by propionate, a downstream metabolite of propane oxidation (Doughty et al, 2006). Moreover, propionate acts as a repressor of alkane degradation in P. butanovora by competitive inhibition for the BMO catalytic site (Doughty et al, 2007). It has been shown that expression of BMO-encoding genes is activated by the putative sigma (54)-transcriptional regulator BmoR.…”

Tailoring the Morphology of g‐C3N4 by Self‐Assembly towards High Photocatalytic Performance

“…The mechanism of action of propionate, another BMO inhibitor, was studied using these mutants. It was shown that both reversible inhibition and inactivation of BMO occurred with propionate because some but not all activity could be recovered by washing of the cells to remove the propionate 47c. Although reversible inhibition was seen with all mutants tested, only those mutants close to the active site had any effect on the irreversible inactivation of BMO by propionate.…”

“…Irreversible inactivation of the BMO enzyme by propionate may be via coordination of the aliphatic tail of propionate to Fe ions at the active site, resulting in the uncoupling of O 2 activation from substrate oxidation and leading to the formation of H 2 O 2 , which damages the enzyme. Interestingly, sMMO is not inactivated by propionate and so this is another MMO‐like phenotype displayed by the G113N mutant 47c…”