Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C2–C9 alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C1–C5 alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by k cat : K m ratios). Relative rates of oxidation for C1–C5 alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate K m values. The low micromolar K m for linear C2–C5 alkanes and the millimolar K m for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar K m and k cat values to those of methane. This inability to kinetically discriminate between the C1 alkane and C1 alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C2–C5 do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C3–C5 alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes ≥C2, despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMMO.

Evidence for Involvement of Copper Ions and Redox State in Regulation of Butane Monooxygenase in Pseudomonas butanovora

ABSTRACT Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C2-C9 alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of β-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O2 when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH4Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O2 (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O2, CuSO4 (0.5 μM) repressed 1-butanol-dependent induction of β-galactosidase activity. Under oxic conditions (20% O2 [vol/vol]), significantly higher concentrations of CuSO4 (2 μM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu2+ reducing agent Na ascorbate (100 μM) and CuSO4 (0.5 μM) repressed the induction of β-galactosidase activity under oxic conditions to the same extent that 0.5 μM CuSO4 alone repressed it under anoxic conditions. Under oxic conditions, 2 μM CuSO4 repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO4 repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

Evidence for Modified Mechanisms of Chloroethene Oxidation in Pseudomonas butanovora Mutants Containing Single Amino Acid Substitutions in the Hydroxylase α-Subunit of Butane Monooxygenase

ABSTRACT The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the α-subunit of butane monooxygenase hydroxylase (BMOH-α) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O2 uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-α mutants might allow insights into the catalytic mechanism of BMO to be obtained.

Site-Directed Amino Acid Substitutions in the Hydroxylase α Subunit of Butane Monooxygenase from Pseudomonas butanovora: Implications for Substrates Knocking at the Gate

ABSTRACT Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase α subunit of butane monooxygenase (BMOH-α) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-α were altered to the corresponding residues from the α subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 μM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 μM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-α.

Product Repression of Alkane Monooxygenase Expression in Pseudomonas butanovora

ABSTRACT Physiological and regulatory mechanisms that allow the alkane-oxidizing bacterium Pseudomonas butanovora to consume C2 to C8 alkane substrates via butane monooxygenase (BMO) were examined. Striking differences were observed in response to even- versus odd-chain-length alkanes. Propionate, the downstream product of propane oxidation and of the oxidation of other odd-chain-length alkanes following β-oxidation, was a potent repressor of BMO expression. The transcriptional activity of the BMO promoter was reduced with as little as 10 μM propionate, even in the presence of appropriate inducers. Propionate accumulated stoichiometrically when 1-propanol and propionaldehyde were added to butane- and ethane-grown cells, indicating that propionate catabolism was inactive during growth on even-chain-length alkanes. In contrast, propionate consumption was induced (about 80 nmol propionate consumed · min−1 · mg protein−1) following growth on the odd-chain-length alkanes, propane and pentane. The induction of propionate consumption could be brought on by the addition of propionate or pentanoate to the growth medium. In a reporter strain of P. butanovora in which the BMO promoter controls β-galactosidase expression, only even-chain-length alcohols (C2 to C8) induced β-galactosidase following growth on acetate or butyrate. In contrast, both even- and odd-chain-length alcohols (C3 to C7) were able to induce β-galactosidase following the induction of propionate consumption by propionate or pentanoate.

Cometabolic transformation of cis-1,2-dichloroethylene and cis-1,2-dichloroethylene epoxide by a butane-grown mixed culture

Aerobic cometabolism of cis-1,2-dichloroethylene (c-DCE) by a butane-grown mixed culture was evaluated in batch kinetic tests. The transformation of c-DCE resulted in the coincident generation of c-DCE epoxide. Chloride release studies showed ∼75% oxidative dechlorination of c-DCE. Mass spectrometry confirmed the presence of a compound with mass-to-charge-fragment ratios of 112, 83, 48, and 35. These values are in agreement with the spectra of chemically synthesized c-DCE epoxide. The transformation of c-DCE required O2, was inhibited by butane and was inactivated by acetylene (a known monooxygenase inactivator), indicating that a butane monooxygenase enzyme was likely involved in the transformation of c-DCE. This study showed c-DCE epoxide was biologically transformed, likely by a butane monooxygenase enzyme. c-DCE epoxide transformation was inhibited by both acetylene and c-DCE indicating a monooxygenase enzyme was involved. The epoxide transformation was also stopped when mercuric chloride (HgCl2) was added as a biological inhibitor, further support a biological transformation. To our knowledge this is the first report of the biological transform c-DCE epoxide by a butane-grown culture.

Effects of Dichloroethene Isomers on the Induction and Activity of Butane Monooxygenase in the Alkane-Oxidizing Bacterium “Pseudomonas butanovora”

ABSTRACT We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium “Pseudomonas butanovora.” Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ∼20 nmol mg protein−1 of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein−1. Oxidation of similar amounts of each DCE isomer (∼20 nmol mg protein−1) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown “P. butanovora” did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.