Scott A. Ensign

Alkene Monooxygenase from Xanthobacter Strain Py2 PURIFICATION AND CHARACTERIZATION OF A FOUR-COMPONENT SYSTEM CENTRAL TO THE BACTERIAL METABOLISM OF ALIPHATIC ALKENES

Alkene monooxygenase fromXanthobacter strain Py2 is an inducible enzyme that catalyzes the O2- and NADH-dependent epoxidation of short chain (C2 to C6) alkenes to their corresponding epoxides as the initial step in the utilization of aliphatic alkenes as carbon and energy sources. In the present study, alkene monooxygenase has been resolved from the soluble fraction of cell-free extracts into four components, each of which has been purified to homogeneity, that are obligately required for alkene epoxidation activity. The four required components are 1) a monomeric 35.5-kDa protein containing 1 mol of FAD and a probable 2Fe-2S center; 2) a 13.3-kDa ferredoxin containing a Rieske-type 2Fe-2S cluster; 3) an 11-kDa monomeric protein that contains no detectable cofactors; and 4) a 212-kDa α2β2γ2 multimeric protein containing four atoms of nonheme iron. The 35.5-kDa protein has been characterized as an NADH reductase. The physiological electron acceptor for the reductase was the Rieske-type ferredoxin, which is proposed to be an intermediate electron carrier between the reductase and terminal catalytic component of the system. The 212-kDa protein was specifically inactivated in cell-free extracts by the mechanism-based inactivator propyne, suggesting that it is the catalytic component and contains the active site(s) for O2 activation and alkene epoxidation. The subunit structure and metal analysis of this component suggest that it contains two diiron centers, one for each αβγ protomeric unit. No specific enzymatic activities could be assigned for the 11-kDa protein, but this component was obligately required for steady-state alkene epoxidation. The alkene monooxygenase components were expressed during growth of Xanthobacter Py2 on aliphatic alkenes or epoxides and repressed during growth on other carbon sources. The electron transfer components of alkene monooxygenase were highly specific: other reductase activities present in Xanthobacter were incapable of transferring electrons to the Rieske-type ferredoxin or substituting for the reductase in the alkene monooxygenase complex. Likewise, other bacterial and plant ferredoxins were unable to substitute for the Rieske-type ferredoxin in mediating electron transfer to the oxygenase. The biochemical properties of alkene monooxygenase described in this study suggest that this enzyme combines elements of both the well-characterized aromatic dioxygenase (two-component electron transfer scheme) and methane monooxygenase (small regulatory protein and diiron oxygenase) multicomponent enzyme systems.

New roles for CO2 in the microbial metabolism of aliphatic epoxides and ketones.

Abstract Short-chain aliphatic epoxides and ketones are two classes of toxic organic compounds formed biogenically and anthropogenically. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds (e.g., alkenes, alkanes, and secondary alcohols) by a number of diverse bacteria. One bacterium capable of using both classes of compounds is the gram-negative aerobe Xanthobacter strain Py2. Studies of epoxide and ketone (acetone) metabolism by Xanthobacter strain Py2 have revealed a central role for CO2 in these processes. Both classes of compounds are metabolized by carboxylation reactions that produce β-keto acids as products. The epoxide- and ketone-converting enzymes are distinct carboxylases with molecular properties and cofactor requirements unprecedented for other carboxylases. Epoxide carboxylase is a four-component multienzyme complex that requires NADPH and NAD+ as cofactors. In the course of epoxide carboxylation, a transhydrogenation reaction occurs wherein NADPH undergoes oxidation and NAD+ undergoes reduction. Acetone carboxylase is a multimeric (three-subunit) ATP-dependent enzyme that forms AMP and inorganic phosphate as ATP hydrolysis products in the course of acetone carboxylation. Recent studies have demonstrated that acetone metabolism in diverse anaerobic bacteria (sulfate reducers, denitrifiers, phototrophs, and fermenters) also proceeds by carboxylation reactions. ATP-dependent acetone carboxylase activity has been demonstrated in cell-free extracts of the anaerobic acetone-utilizers Rhodobacter capsulatus, Rhodomicrobium vannielii, and Thiosphaera pantotropha. These studies have identified new roles for CO2 as a cosubstrate in the metabolism of two classes of important xenobiotic compounds. In addition, two new classes of carboxylases have been identified, the investigation of which promises to reveal new insights into biological strategies for the fixation of CO2 to organic substrates.

Biochemical, Molecular, and Genetic Analyses of the Acetone Carboxylases from Xanthobacter autotrophicus Strain Py2 and Rhodobacter capsulatus Strain B10

Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO(2) to form acetoacetate. In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus was purified to homogeneity and compared to that of Xanthobacter autotrophicus strain Py2, the only other organism from which an acetone carboxylase has been purified. The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (alpha(2)beta(2)gamma(2) multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH(3)COCH(3) + CO(2) + ATP-->CH(3)COCH(2)COO(-) + H(+) + AMP + 2P(i)), and kinetic properties (K(m) for acetone, 8 microM; k(cat) = 45 min(-1)). Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources. The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of X. autotrophicus and sequence analysis of the R. capsulatus genome and were found to be clustered in similar operons consisting of the genes acxA (beta subunit), acxB (alpha subunit), and acxC (gamma subunit). Transposon mutagenesis of X. autotrophicus revealed a requirement of sigma(54) and a sigma(54)-dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression. A potential sigma(54)-dependent promoter 122 bp upstream of X. autotrophicus acxABC was identified. An AcxR gene homolog was identified 127 bp upstream of acxA in R. capsulatus, but this activator lacked key features of sigma(54)-dependent activators, and the associated acxABC lacked an apparent sigma(54)-dependent promoter, suggesting that sigma(54) is not required for expression of acxABC in R. capsulatus. These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene expression in gram-negative acetone-utilizing bacteria.

Purification to Homogeneity and Reconstitution of the Individual Components of the Epoxide Carboxylase Multiprotein Enzyme Complex fromXanthobacter Strain Py2

Epoxide metabolism in the aerobic bacteriumXanthobacter strain Py2 proceeds by an NADPH- and NAD+-dependent carboxylation reaction that forms β-keto acids as products. Epoxide carboxylase, the enzyme catalyzing this reaction, was resolved from the soluble fraction of cell-free extracts into four protein components that are obligately required for functional reconstitution of epoxide carboxylase activity. One of these components, component II, has previously been purified and characterized as an NADPH:disulfide oxidoreductase. In the present study, the three additional epoxide carboxylase components have been purified to homogeneity and characterized. These component proteins are as follows: component I, a homohexameric protein consisting of 41.7-kDa subunits; component III, a dimeric protein consisting of 26.0- and 26.2-kDa polypeptides; and component IV, a dimeric protein consisting of a single 25.4-kDa polypeptide. Component I contained 5 mol of tightly bound zinc per mol of protein. Component I was specifically inactivated by methylepoxypropane, a time-dependent irreversible inactivator of epoxide carboxylase activity, suggesting that this component plays an integral role in epoxide binding and activation. No metals or organic cofactors were detected for components III and IV. The molecular weights, N-terminal sequences, and amino acid compositions of the purified epoxide carboxylase components were determined and found to correlate with open reading frames within and adjacent to a cloned fragment of DNA that complementsXanthobacter Py2 mutants defective in epoxide degradation. Using the purified epoxide carboxylase system, epoxide carboxylation was found to be stoichiometrically coupled to the transhydrogenation of pyridine nucleotide cofactors according to the following equation: epoxypropane + CO2 + NADPH + NAD+ → acetoacetate + H+ + NADP+ + NADH.

Evidence that a linear megaplasmid encodes enzymes of aliphatic alkene and epoxide metabolism and coenzyme M (2-mercaptoethanesulfonate) biosynthesis in Xanthobacter strain Py2.

The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by a sequence of three reactions resulting in epoxide ring opening and carboxylation to form acetoacetate. Coenzyme M (2-mercaptoethanesulfonic acid) (CoM) plays a central role in epoxide carboxylation by serving as the nucleophile for epoxide ring opening and the carrier of the C(3) unit that is ultimately carboxylated to acetoacetate, releasing CoM. In the present work, a 320-kb linear megaplasmid has been identified in the gram-negative bacterium Xanthobacter strain Py2, which contains the genes encoding the key enzymes of propylene oxidation and epoxide carboxylation. Repeated subculturing of Xanthobacter strain Py2 under nonselective conditions, i.e., with glucose or acetate as the carbon source in the absence of propylene, resulted in the loss of the propylene-positive phenotype. The propylene-negative phenotype correlated with the loss of the 320-kb linear megaplasmid, loss of induction and expression of alkene monooxgenase and epoxide carboxylation enzyme activities, and the loss of CoM biosynthetic capability. Sequence analysis of a hypothetical protein (XecG), encoded by a gene located downstream of the genes for the four enzymes of epoxide carboxylation, revealed a high degree of sequence identity with proteins of as-yet unassigned functions in the methanogenic archaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii and in Bacillus subtilis. The M. jannaschii homolog of XecG, MJ0255, is located next to a gene, MJ0256, that has been shown to encode a key enzyme of CoM biosynthesis (M. Graupner, H. Xu, and R. H. White, J. Bacteriol. 182: 4862-4867, 2000). We propose that the propylene-positive phenotype of Xanthobacter strain Py2 is dependent on the selective maintenance of a linear megaplasmid containing the genes for the key enzymes of alkene oxidation, epoxide carboxylation, and CoM biosynthesis.

Revisiting the glyoxylate cycle: alternate pathways for microbial acetate assimilation

The glyoxylate cycle, identified by Kornberg et al. in 1957, provides a simple and efficient strategy for converting acetyl‐CoA into anapleurotic and gluconeogenic compounds. Studies of a number of bacteria capable of growth with C2 compounds as the sole carbon source have revealed that they lack the key glyoxylate cycle enzyme isocitrate lyase, suggesting that alternative pathway(s) for acetate assimilation exist in these bacteria. Recent studies of acetate assimilation in methylotrophs and purple phototrophs have revealed remarkable and complex new pathways for assimilation of acetate in the absence of isocitrate lyase. The details of these new pathways are the subject of this MicroCommentary.

Heterologous expression of bacterial Epoxyalkane:Coenzyme M transferase and inducible coenzyme M biosynthesis in Xanthobacter strain Py2 and Rhodococcus rhodochrous B276.

Coenzyme M (CoM) (2-mercaptoethanesulfonic acid) biosynthesis is shown to be coordinately regulated with the expression of the enzymes of alkene and epoxide metabolism in the propylene-oxidizing bacteria Xanthobacter strain Py2 and Rhodococcus rhodochrous strain B276. These results provide the first evidence for the involvement of CoM in propylene metabolism by R. rhodochrous and demonstrate for the first time the inducible nature of eubacterial CoM biosynthesis.

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

SUMMARY Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO2 to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO2 to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.

Photoheterotrophic Metabolism of Acrylamide by a Newly Isolated Strain of Rhodopseudomonas palustris

ABSTRACT Acrylamide, a neurotoxin and suspected carcinogen, is produced by industrial processes and during the heating of foods. In this study, the microbial diversity of acrylamide metabolism has been expanded through the isolation and characterization of a new strain of Rhodopseudomonas palustris capable of growth with acrylamide under photoheterotrophic conditions. The newly isolated strain grew rapidly with acrylamide under photoheterotrophic conditions (doubling time of 10 to 12 h) but poorly under anaerobic dark or aerobic conditions. Acrylamide was rapidly deamidated to acrylate by strain Ac1, and the subsequent degradation of acrylate was the rate-limiting reaction in cell growth. Acrylamide metabolism by succinate-grown cultures occurred only after a lag period, and the induction of acrylamide-degrading activity was prevented by the presence of protein or RNA synthesis inhibitors. 13C nuclear magnetic resonance studies of [1,2,3-13C]acrylamide metabolism by actively growing cultures confirmed the rapid conversion of acrylamide to acrylate but failed to detect any subsequent intermediates of acrylate degradation. Using concentrated cell suspensions containing natural abundance succinate as an additional carbon source, [13C]acrylate consumption occurred with the production and then degradation of [13C]propionate. Although R. palustris strain Ac1 grew well and with comparable doubling times for each of acrylamide, acrylate, and propionate, R. palustris strain CGA009 was incapable of significant acrylamide- or acrylate-dependent growth over the same time course, but grew comparably with propionate. These results provide the first demonstration of anaerobic photoheterotrophic bacterial acrylamide catabolism and provide evidence for a new pathway for acrylate catabolism involving propionate as an intermediate.

Characterization of 2-Bromoethanesulfonate as a Selective Inhibitor of the Coenzyme M-Dependent Pathway and Enzymes of Bacterial Aliphatic Epoxide Metabolism

Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO(2) fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO(2)-fixing enzyme 2-KPCC.