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1 Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA
2 Program of Molecular and Cellular Biology, Oregon State University, Corvallis, OR 97331-3804, USA
3 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331-3804, USA
4 Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331-3804, USA
Correspondence
Peter J. Bottomley
Peter.Bottomley{at}oregonstate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Lipscomb, 1994; Smith et al., 2003; Smith & Dalton, 2004). In this connection we have studied the transcriptional and physiological regulation of butane monooxygenase (BMO) in ‘Pseudomonas butanovora’. BMO oxidizes n-alkanes C2 to C9 to alcohols, which are subsequently metabolized to fatty acids (Arp, 1999; Doughty et al., 2006). We have shown that the activity of the bmo promoter is induced by alcohol, aldehyde and epoxide products of alkane and alkene oxidation, and repressed by micromolar concentrations of propionate (Doughty et al., 2005, 2006; Sayavedra-Soto et al., 2001, 2005). Furthermore, although the organic acids acetate, propionate, butyrate and lactate can act as exogenous sources of reductant to stimulate BMO activity, propionate and butyrate do not sustain linear rates of BMO activity for as long as equivalent amounts of lactate or acetate (Doughty et al., 2005). In this study our observations were extended to show that propionate and butyrate inactivate pre-existing BMO.
BMO shares a high level of amino acid sequence identity with the well-characterized soluble methane monooxygenase (sMMO) found in methanotrophic bacteria (Sluis et al., 2002). Inhibitors and inactivators of sMMO include (i) suicide substrates such as terminal alkynes, which covalently bind to active-site residues following a catalytic transformation in the enzyme's active site (Prior & Dalton, 1985); (ii) heavy metals (e.g. Cu2+) that rapidly and irreversibly inhibit the reductase subunit of the enzyme, preventing electron transfer to the hydroxylase subunit (Green et al., 1985; Jahng & Wood, 1996); and (iii) H2O2, which inactivates the enzyme through an unknown mechanism (Astier et al., 2003). Because propionate lacked structural similarity to any of the known inhibitors and inactivators of sMMO mentioned above, we thought it worthwhile to characterize the effect of propionate on BMO. As the project evolved, it became clear that intermediate chain-length fatty acids might be potentially useful for further studies focused on dissecting the catalytic mechanism of BMO.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hamamura et al., 1999; Vangnai & Arp, 2001). Cells were harvested by sedimentation in a centrifuge (6500 g for 10 min), and washed three times in 50 mM sodium/potassium phosphate buffer, pH 7.1. Lactate-supported, ethene-dependent ethene oxide production is a convenient measure of BMO activity. Ethene oxide assays were performed as described previously (Doughty et al., 2005) except that 0.5 ml samples of the headspace were taken at 2.5 min intervals and injected into separate 7 ml sealed vials for later analysis. This variation of the assay allowed for the prompt sampling of a large number of vials and separated experimental execution from the analysis of gas samples. Protein concentrations were determined using the micro-biuret assay described previously (Gornall et al., 1949).
In vivo inactivation of BMO in ‘P. butanovora’.
Butane-grown cultures of ‘P. butanovora’ were harvested and washed as described above. Vials (160 ml) containing 80 ml of cells (OD600 0.5; 0.2 mg protein ml–1), resuspended in 50 mM sodium/potassium phosphate buffer, served as the reaction chambers for the assays. Vials received 10 mM lactate as an exogenous reductant to support BMO activity and 10 mM of either propionate, butyrate or acetate. Assay mixtures were shaken on an orbital shaker at 200 r.p.m. and 30 °C. Accurate assessment of time-dependent inactivation of BMO required the prompt interruption of the assay. To accomplish this, aliquots of cells were injected into ‘stop vials’ (160 ml) containing 10 ml 50 mM sodium/potassium phosphate buffer in equilibrium with headspace containing 0.4 mmol butane gas (approx. 200 µM aqueous concentration), which prevented further inactivation of the BMO enzyme during the washing procedure. Cells were washed three times by centrifugation (6500 g for 10 min), resuspended in 50 mM sodium/potassium phosphate buffer, and tested for residual BMO activity using the lactate-dependent ethene oxidation assay described above.
Because substrate oxidation by BMO is dependent upon O2 as a co-substrate, we also examined the effect of propionate on BMO under anoxic conditions. Vials (160 ml) containing 30 ml of 50 mM sodium/potassium phosphate buffer were sealed with butyl rubber stoppers and made anoxic by flushing the headspace with argon for 2 min. Stock solutions of lactate and propionate, as well as cell suspensions, were prepared using the same method. Lactate (10 mM), propionate (10 mM) and cell suspensions were injected into the reaction vial with an airtight syringe. Aliquots were removed from each treatment and injected into ‘stop’ vials, washed three times, and tested for BMO activity as described earlier.
Investigation of propionate-dependent inactivation in mutant strains of ‘P. butanovora’ with modified BMO hydroxylase.
Amino acid sequence alignments were used to compared the hydroxylase subunit of BMO with the hydroxylase subunit (MMOH) of the well-characterized sMMO enzymes of Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b (Halsey et al. 2006). Amino acid residues were identified that were likely associated with (a) the active site, (b) entry of substrate into the active site, or (c) the interaction between the hydroxylase subunit and a regulatory subunit, BmoB. These amino acid residues were targeted for site-directed mutagenesis and are described in Table 1. The mutants were screened for sensitivity to propionate as described above.
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), and [2-14C]propionate [50 mCi mmol–1 (1.8 GBq mmol–1)] was purchased from American Radiolabelled Chemicals. Butane-grown cells were harvested and washed with 50 mM sodium/potassium phosphate buffer containing 50 µg chloramphenicol ml–1 to stop the synthesis of protein. Vials (2 ml) containing 200 µl of resuspended cells (approx. 140 µg protein) received 10 mM lactate to serve as an electron donor for BMO activity and 0.2 µmol (1 mM) of either [2-14C]propionate or 14C2H2. All treatments were incubated for 30 min in a covered water bath shaker (30 °C and 150 r.p.m.). Following incubation, cells were washed three times in 50 mM sodium/potassium phosphate buffer and lysed by the addition of SDS and DTT (Sambrook et al., 1989). Protein (20 µg) from each treatment was loaded onto a 12.5 % SDS-PAGE gel and resolved at 15 mA. SDS-PAGE gels were stained with Coomassie blue to visualize the protein, destained and dried in a vacuum desiccator. The incorporation of 14C label was visualized by phosphorimaging and the relative signal densities determined with the ImageQuant software provided by the manufacturer (Molecular Dynamics). A standard curve was prepared by diluting different amounts of 14C2H2-labelled cell extracts into unlabelled cells extracts and comparing the intensities of the images obtained from the standards with the 14C-labelled experimental treatments.
Because complete inactivation of BMO did not occur following incubation with propionate, an experiment was carried out to discriminate between inactivation of a fraction of BMO versus a reduced turnover rate by all BMO molecules. The effect of propionate inactivation on 14C2H2 label incorporation into BMO was examined. Butane-grown cells, washed and treated as described above, were exposed to either 10 mM lactate or 10 mM lactate plus 10 mM propionate for 2 h. Cells were washed and aliquots of each treatment were tested for BMO activity or incubated with 10 mM lactate plus 14C2H2 for 2 h. Following incubation with 14C2H2, whole-cell protein was examined by SDS-PAGE and the incorporation of 14C into the -subunit polypeptide of BMO hydroxylase determined as described above.
Propionate inactivation of monooxygenases from other bacteria.
Burkholderia cepacia G4 and Pseudomonas mendocina KR-1 were grown in the same medium as described for ‘P. butanovora’ with toluene (1 mM) as the carbon source. Cultures were shaken at 200 r.p.m. on an orbital shaker at 30 °C. The methanotrophic bacteria Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), were grown under copper-limiting conditions in a KNO3 minimal salts medium to promote the production of sMMO rather than pMMO (Lee et al., 2006; Whittenbury et al., 1970). M. capsulatus (Bath) was incubated at 37 °C and M. trichosporium OB3b was incubated at 30 °C on an orbital shaker at 200 r.p.m. Monooxygenase activity was monitored with the ethene oxide assay described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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showed previously that acetate, propionate and butyrate are effective exogenous sources of reductant for supporting BMO activity in propane-grown cells of ‘P. butanovora’. In this work it can be clearly seen that propionate (10 mM) or butyrate (10 mM) quickly and effectively reduced lactate-dependent BMO activity (Fig. 1). In contrast, the rate of lactate-dependent BMO activity was not affected when acetate (10 mM) or an equivalent volume of phosphate buffer was added to the assay. The kinetics of propionate inactivation of BMO were unchanged even at concentrations as low as 0.1 mM, suggesting that the effect of propionate on BMO reaches saturation at concentrations below 0.1 mM. The same organic acids were tested to determine if the BMO inactivation was reversible or irreversible (Fig. 2). Propionate and butyrate irreversibly inactivated BMO in a time-dependent manner, and BMO activity was not completely restored when propionate or butyrate were removed (Fig. 2). In contrast, BMO activity did not decrease in cells treated with either lactate or a combination of lactate and acetate. From data in Fig. 1 it was calculated that propionate had reduced BMO activity by 73–80 % of its initial rate between 5 and 7.5 min after propionate addition. In contrast, Fig. 2 shows that only 40 % of BMO was irreversibly inactivated following a similar length of incubation with propionate. These data suggest that propionate acts as both a reversible inhibitor and irreversible inactivator of BMO. The remainder of this study focused on the irreversible effect of propionate on BMO activity.
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). Because BMO hydroxylation of butane is dependent upon O2 as a co-substrate we thought it worthwhile to determine if O2 was required for propionate-dependent BMO inactivation. Under anoxic conditions, propionate (10 mM) failed to inactivate BMO (Fig. 3b). In contrast, when 22 nmol O2 was supplied to anoxic cells, the ability of propionate to inactivate BMO was restored. Control treatments which received either no O2, or O2 and no propionate, did not show a significant decrease in BMO activity. Propionate-inactivated BMO did not regain activity following subsequent incubation under anoxic conditions, indicating that BMO activity was protected by anoxia, rather than restored during anoxic treatment. The O2-dependence of inactivation suggests that the catalytic cycle of BMO is involved in propionate-dependent inactivation.
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[2-14C]Propionate and 14C2H2 labelling experiments
The possibility that propionate might covalently bind to BMO during inactivation was explored with [2-14C]propionate. Although SDS-PAGE analysis of cellular proteins following treatment with [2-14C]propionate showed weak labelling of a few polypeptides, the molecular masses of 14C-labelled bands did not correspond to the molecular masses of BMO polypeptides. The possibility was considered that propionate might label BMO in an SDS-labile manner. When cell extracts were electrophoresed on native polyacrylamide gels, they also failed to show any association of [2-14C]propionate with polypeptides of molecular masses equivalent to BMO hydroxylase or reductase (data not shown). These data suggest that [2-14C]propionate did not covalently bind to the BMO enzyme, and that propionate inactivation did not follow the model of a classical monooxygenase suicide substrate such as acetylene. Furthermore, we explored the possibility that propionate was oxidized by BMO. Propionate consumption by butane-grown ‘P. butanovora’ was unaffected by acetylene within the limits of detection [(5 nmol propionate min–1 (mg protein)–1)]. These data further suggest that propionate is not a substrate of BMO.
Because propionate did not completely inactivate BMO, we attempted to distinguish between reduced BMO activity being due to inactivation of a fraction of BMO molecules versus a reduced rate of turnover of all BMO molecules. Butane-grown cells incubated with propionate for 2 h retained 
10 % of BMO activity, and all remaining BMO activity was found to be acetylene sensitive (Fig. 4a, b). Furthermore, the incorporation of 14C from 14C2H2 into the 58 kDa polypeptide of the -subunit of BMO hydroxylase was reduced by 90 % relative to the buffer control, and was consistent with the propionate-dependent reduction of BMO activity (Fig. 4c). These data suggest that propionate caused BMO inactivation rather than an overall reduction in the rate of BMO turnover.
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-subunit of the BMO hydroxylase-subunit of BMO hydroxylase (BMOH-) were examined for propionate-dependent inactivation of BMO (Table 2). The effects of propionate on BMO differed among the mutant strains. For example, in the case of the G113N mutant, in which a glycine was replaced by an asparagine residue in a region adjacent to, or contributing to, the active site, BMO activity was not inactivated by propionate. In contrast, mutant strain T148C, in which a threonine in the active-site region was replaced by a cysteine, showed a significant increase in propionate-dependent inactivation of BMO relative to wild-type. Mutants F321Y and L279F, which were modified at amino acid positions believed to be involved in the interaction of BMOH- with BMOB, were inactivated by propionate to a similar extent as wild-type. Interestingly, reversible inhibition of BMO was observed in all mutant strains, including the G113N mutant. Inhibition of BMO activity by propionate in the G113N mutant was time-dependent, and a 10 min incubation with propionate was sufficient to inhibit 50 % of BMO activity. Propionate-treated G113N mutant regained BMO activity equivalent to the untreated cells following the washing procedure.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-subunit of BMO hydroxylase) was changed to an asparagine residue. Because the reversible inhibition by propionate remained unaffected in this mutant, these data indicated that reversible inhibition and irreversible inactivation of BMO occur through separate mechanisms.
Propionate inhibition of BMO ties in well with the recent discovery that propionate represses the transcription of BMO genes (Doughty et al., 2006). We previously discussed a model of BMO transcriptional regulation to explain how fatty acid products of alkane oxidation might repress BMO. This form of feedback inhibition resembles the regulation of lipid synthesis (Fab) and fatty acid oxidation (Fad) characterized in Escherichia coli (Cronan & Subrahmanyam, 1998). In this connection it is interesting to note that three Fab enzymes are not only repressed by long-chain acyl-CoAs at the level of transcription, but also controlled post-translationally through feedback inhibition by acylated acyl-carrier proteins (acyl-ACP) (Heath & Rock, 1996; Marrakchi et al., 2002). The net result is close coupling of the reductant-consuming lipid biosynthesis pathway with reductant-generating fatty acid 
-oxidation. Similarly, the inhibition of alkane oxidation by the downstream products of BMO activity could prevent the allocation of reductant to BMO under circumstances where the production of fatty acids via alkane oxidation exceeds the carbon and energy requirements of the cell. A physiological role for inactivation of BMO can also be envisaged, particularly if the monooxygenase turns over slowly, generates toxic H2O2 in the absence of substrate, and also continues to consume reductant.
Because we could not detect propionate consumption during BMO inactivation, and [2-14C]propionate label did not associate with the BMO polypeptides after electrophoresis under denaturing conditions, propionate inactivation did not follow the model of a monooxygenase suicide substrate such as acetylene. In the context of existing literature on sMMO, two possible mechanisms for propionate-dependent inactivation of BMO can be proposed. First, in vitro studies showed that when sMMO hydroxylase is electrically reduced at the surface of an electrode, O2 is reduced to H2O2 by the hydroxylase and subsequently inactivates it (Astier et al., 2003). We propose a model of BMO inactivation in which propionate stimulates the production of H2O2 by BMO and causes oxidative enzyme damage. Propionate-dependent formation of H2O2 by BMO would reconcile our observations that BMO inactivation is O2- and turnover-dependent, while propionate oxidation per se does not occur. A model based upon the catalytic cycle of sMMO is shown in Fig. 5 (Brazeau et al., 2001; Lee et al., 1993a, b; Liu et al., 1995; Zhang & Lipscomb, 2006). The diferric active site, shown at the bottom of the model, is reduced and used to break one bond of O2, forming intermediate P. The scission of the second O2 bond results in the formation of intermediate Q. Alternatively, the reaction of the reduced form of sMMO with O2 can result in H2O2 formation and a return of sMMO to a diferric resting state (Zhang & Lipscomb, 2006). Although the presence of the regulatory subunit (MMOB) eliminates the production of H2O2 by sMMO hydroxylase, H2O2 production resumed, as did enzyme inactivation, following the introduction of substrate to the reaction mixture (Astier et al., 2003). In this context, the aliphatic tail of propionate might resemble the alkane substrate of BMO sufficiently well to reduce the coupling of O2 activation to substrate oxidation and result in H2O2 formation. In this connection there is some indication that BmoB is not involved in the coupling of O2 activation to substrate turnover by BMO (Dubbels et al., 2007). In methane monooxygenase hydroxlyase (MMOH), multiple substrate-binding sites have been proposed for substrates larger than methane (Sazinsky & Lippard, 2005), raising the possibility that butane and propionate compete for a site outside the substrate-binding pocket. Conceptually, this could reconcile our observations that (i) butane protects BMO from propionate and (ii) propionate is not oxidized by BMO (as propionate does not enter the active site). Our results, although preliminary, raise the possibility that alternative substrate-binding pockets have replaced the role of the coupling subunit in substrate recognition. Second, propionate may form an ionic bond with the active-site irons, and inactivate the enzyme by blocking the active site. In this context, it is interesting to note that crystallographic data obtained from the diferric form of sMMO showed an unidentified electron density within the hydrophobic substrate-binding pocket. Researchers tentatively identified this structure as acetate, a component of the crystallization buffer, with the carboxyl group of acetate forming a bridging ligand between the diirons (Rosenzweig et al., 1997). Additionally, recent research on a structurally related diiron desaturase used acetate as a molecular mimic for O2 binding. The authors suggested that acetate produced a carboxyl shift in a glutamate residue, resulting in a change in coordination of an active-site iron (Moche et al., 2003). Interestingly, a similar change was observed following the binding of substrate (acyl-ACP) and it is hypothesized that the observed change in iron coordination increases the reactivity of the active site with O2 (Moche et al., 2003). Our research, although preliminary and in vivo, raises the possibility that propionate could be useful as a probe to uncover some of the mechanistic details of BMO. Because of the novelty of propionate-dependent inactivation, we extended our study to mutant strains of ‘P. butanovora’ in which single amino acid substitutions had been made to the -subunit of the hydroxylase (Halsey et al., 2006). Because amino acids G113 and T148 are predicted to be close to the enzyme active site (Table 1
), the altered outcome of propionate inactivation observed in mutant strains G113N and T148C suggests that propionate enters the active site of BMO. Although it remains unclear why mutant strains G113N and T148C display altered propionate-dependent inactivation relative to wild-type, two possibilities can be discussed. First, structural prediction programs indicate that the size of the hydrophobic substrate-binding cavity of BMO will be decreased by the G
N mutation and increased by the TC mutation (Halsey et al., 2006). In this context, the insensitivity of mutant strain G113N to propionate may indicate that the active site of the enzyme is inaccessible to propionate. In contrast, the larger substrate-binding cavity of T148C may increase the accessibility of the active site to propionate. Second, the altered outcome of propionate-dependent inactivation in the mutant strains might reflect a subtle change in the tendency of these enzymes to release H2O2. Indeed, recent research on a structurally related diiron desaturase indicated that a mutant form of the enzyme containing a single amino acid substitution near the active site produced 40-fold more H2O2 than the wild-type enzyme (Guy et al., 2006). Clearly, we are in a position to move forward and obtain crystal structures of BMO from both the wild-type and mutant strains, and carry out in vitro studies with the recently purified BMO (Dubbels et al. 2007) to gain a better understanding of short-chain fatty acid inactivation.
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ACKNOWLEDGEMENTS |
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Edited by: J. A. Vorholt
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 26 March 2007;
revised 27 June 2007;
accepted 24 July 2007.
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), propionate (
) or butyrate (
), or an equivalent volume (10 µl) of buffer (
). Data points are the means of three replicates and error bars represent the standard deviation of the means.
