Alkenes and ketones are two classes of ubiquitous, toxic organic compounds in natural environments produced in several biological and anthropogenic processes. 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 by many diverse bacteria. The aerobic metabolism of some of the smallest and most volatile of these compounds (propylene, acetone, isopropanol) involves novel carboxylation reactions resulting in a common product acetoacetate. Propylene is metabolized in a four-step pathway involving five enzymes where the penultimate step is a carboxylation reaction catalyzed by a unique disulfide oxidoreductase that couples reductive cleavage of a thioether linkage with carboxylation to produce acetoacetate. The carboxylation of isopropanol begins with conversion to acetone via an alcohol dehydrogenase. Acetone is converted to acetoacetate in a single step by an acetone carboxylase which couples the hydrolysis of MgATP to the activation of both acetone and bicarbonate, generating highly reactive intermediates that are condensed into acetoacetate at a Mn2+ containing the active site. Acetoacetate is then utilized in central metabolism where it is readily converted to acetyl-coenzyme A and subsequently converted into biomass or utilized in energy metabolism via the tricarboxylic acid cycle. This review summarizes recent structural and biochemical findings that have contributed significant insights into the mechanism of these two unique carboxylating enzymes.
Alkene and ketone metabolism
Considerable attention has been focused on the microbial metabolism of xenobiotic compounds. Two classes of organic compounds of interest in this regard are alkenes and ketones, which are formed by both biogenic and anthropogenic processes. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources by some bacteria and are generated as intermediate metabolites in the metabolism of other compounds by many diverse bacteria.
Alkenes are unsaturated hydrocarbons that contain a carbon–carbon double bond. Many organisms (plant, insect, cyanobacteria) synthesize alkenes naturally for protection against environmental threats [1–3]. Several gaseous and volatile alkenes are produced naturally. The most predominant of these compounds are associated with plants, including ethene [4,5], isoprene , and various monoterpenes. Alkenes have also been reported to be synthesized by various microorganisms [7,8]. For example, the synthesis of alkenes is widespread in cyanobacteria  and are thought to contribute significantly to the hydrocarbon cycle of the upper ocean . Alkenes are additionally an important part of our everyday lives, having numerous industrial uses. They can be used as liquid transportation fuels and as precursors for plastics, where they are obtained by cracking of crude oils . Many unsaturated hydrocarbons, especially the smaller, gaseous alkenes ethene, propene, 1,3-butadiene, and butene, are produced chemically on a large scale . Alkenes serve as a feedstock for the petrochemical industry because they can participate in a wide variety of reactions, predominantly polymerization and alkylation. Due to their reactivity, unbranched aliphatic alkenes (e.g. ethylene, propylene, butene) are of particular concern as potential human health hazards [13,14].
Many organisms synthesize alkenes naturally for protection against environmental threats [1–3]. Several microbial alkene biosynthetic pathways that convert free fatty acids or fatty acids derivatives to alkenes have been identified or reconstructed [15,16]: (a) decarbonylation of fatty aldehydes (AAR-ADO pathway), (b) decarboxylation of fatty acids (OLeTJE pathway), (c) head-to-head’ (i.e. C–C bond forming) hydrocarbon biosynthesis pathway (OleABCD), and (d) polyketide synthase (PKS) pathway (Ols) (Figure 1). The AAR-ADO pathway involves two candidate enzymes: an acyl-ACP reductase (AAR) and an aldehyde-deformylating oxygenase (ADO) [3,17]. In this AAR-ADO pathway, an acyl-ACP in the acyl–acyl carrier protein is reduced to an aldehyde by AAR and then the aldehyde is converted to an alkene with the formation of formate as a co-product by ADO (reaction a, Figure 1). The key enzyme to convert fatty acids to terminal alkenes is named OLeTJE. The fatty acid decarboxylase OleTJE, isolated and purified from Jeotgalicoccus sp. ATCC 8456, is a P450 peroxygenase from the cyp152 family, which directly decarboxylate long-chain FFAs (C16–C20) to α-alkenes using H2O2 as its main source of electrons. It is indicated that OleTJE also includes fatty acid hydroxylase activity and His85 in OleTJE plays an important role in catalysis (reaction b, Figure 1) . The ‘head-to-head’ pathway has been reported to take part in condensation reactions of fatty acid derivatives to generate long-chain alkenes and to require four protein families (OleABCD) (reaction c, Figure 1) . The OleABCD protein families were defined within thiolase, α/β-hydrolase, AMP-dependent ligase/synthase, and short-chain dehydrogenase superfamilies, respectively . The PKS pathway involves a large multi-domain type I polyketide synthase (Ole) (reaction d, Figure 1). Acyl-ACP is loaded to the ACP1 domain by the loading domain (LD), then two carbons from malonyl-CoA are added to the acyl-substrate by the central extension module including ketosynthase, acyltransferase, ketoreductase, and the ACP2 domain and the β-keto group is reduced to a β-hydroxyl. The sulfotransferase domain activates the β-hydroxyl group via sulfonation. Subsequent dehydration and decarboxylation reactions could be catalyzed by the C-terminal thioesterase (TE) domain . Recently, artificial alkene biosynthetic pathways were designed and implemented for the production of alkenes in Escherichia coli. The carboxylic acid reductase (CAR) from Mycobacterium marinum together with the phosphopantetheinyl transferase Sfp from Bacillus subtilis  and fatty acid reductase (FAR) complex encoded by the genes luxC, luxE, and luxD from Photorhabdus luminescens can catalyze the formation of fatty aldehydes from fatty acids [23,24]. Coupled with ADO from cyanobacteria, fatty aldehydes can be converted further to alkenes (reaction e, Figure 1).
Pathways for fatty acid-based alkene biosynthesis.
Many microorganisms, especially bacteria, fungi and yeast, utilize alkenes as sources of carbon and energy. Catabolism of alkenes has traditionally been considered as a strictly oxygen-dependent process. Studies of bacterial alkene-oxidizing metabolism have revealed the involvement of previously unrecognized metabolic pathways, new and surprising types of enzymes, and atypical cofactors . Diverse bacteria, including various strains of Rhodococcus, Mycobacterium, Nocardia, Xanthobacter, Alcaligenes, Pseudomonas, and Corynebacterium, are capable of aerobic growth with short-chain aliphatic alkenes (C2 to C6, e.g. propylene, ethene), aromatic alkenes (e.g. toluene), and chlorinated alkene (vinyl chloride, cis-, trans-1, 2-dichloroethylene, and trichloroethylene) as the sole added carbon source . While some alkene-oxidizing bacteria are restricted to growth on a single alkene, e.g. ethylene or isoprene (2-methylbutadiene), others exhibit less selectivity for the growth substrate. In aerobic, alkene-utilizing bacteria, the initial attack of alkenes always requires molecular oxygen as a co-substrate. The first enzymes in the metabolic pathways of alkanes are monooxygenases, while aromatic hydrocarbons are oxygenated by either mono- or dioxygenases. These enzymes incorporate hydroxyl groups, derived from molecular oxygen, into the aliphatic chain or the aromatic ring. The alcohols formed from aliphatic hydrocarbons are then oxidized to the corresponding acids; the phenolic compounds generated by ring hydroxylation of aromatic hydrocarbons are direct precursors for oxidative ring cleavage [27,28].
More recently, several alkenes have also been demonstrated to be anaerobically utilized as substrates by several species of denitrifying, ferric iron-reducing, and sulfate-reducing bacteria . Anaerobic degradation of straight-chain alkenes by enrichment cultures and sulfate-reducing bacteria has been reported; however, the initial reactions of these unsaturated hydrocarbons in anaerobic bacteria are still unknown. Recent studies reported anaerobic degradation of 1-alkenes by the sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803 isolated from a polluted marine sediment . This strain has been shown to oxidize alkenes into fatty acids at the C-1 double bond (via a primary alcohol), and by the addition of an undefined carbon unit(s) at C-2 and C-3 and at the subterminal carbon of the saturated end of the molecule [C − (ω − 1)] . Finally, the fourth group of anaerobic bacteria, the ‘proton-reducing’ hydrocarbon-degrading bacteria, utilize various alkenes and have been found in syntrophic association with methanogenic archaea [30,31]. These convert alkenes to CO2, H2, and acetate. This disproportionation is thermodynamically feasible only if the steady-state concentration of hydrogen (and possibly that of acetate) is kept at a low level and is achieved by hydrogen and acetate consumption by methanogens.
Ketones are organic compounds that contain a carbonyl group and two aliphatic or aromatic substituents. The substituents may be saturated or unsaturated, linear or branched alkyl groups, or aromatic residues. They may be the same or different. Moreover, the alkyl or aryl substituents can also contain heteroatoms. More than one carbonyl group may be present in a particular molecule . They are widespread in nature including plants, microorganisms, animals, and human, often combined with other functional groups. They are found in several sugars and in natural steroid hormones. Only a few ketones are manufactured on a large scale in the industry. They can be synthesized by a wide variety of methods, and because of their ease of preparation, relative stability, and high reactivity, they are nearly ideal chemical intermediates. Industrially, many complex organic compounds are synthesized using ketones as building blocks in hydrogenation, reductive condensation, reductive amination, oxidation to peroxides, ketal formation, and condensation reactions . Ketones are most widely used as solvents, especially in industries manufacturing explosives, lacquers, paints, and textiles. Ketones are also used in tanning, as preservatives, and in hydraulic fluids. Most commonly used ketones are sugar (ketoses) and the industrial solvent acetone. Acetone is one of the most important industrial solvents used in such familiar products as paints, coatings, and nail-polish removers. They constitute a significant groundwater, air, water, and soil contaminant. Acetone can also be produced biologically by the fermentative metabolism of certain anaerobic bacteria and from ketone body breakdown in mammals [33,34].
Many anaerobic bacteria, such as those in the genus Clostridium, produce acetone as a major fermentation end product . In these bacteria, acetone is formed from the decarboxylation of acetoacetate in reactions catalyzed by acetoacetate decarboxylases (AAD) (reaction a, Figure 2) . In some hydrocarbon-oxidizing bacteria of the genus Mycobacterium, the pathway of propane metabolism involves an initial hydroxylation reaction producing isopropanol, which is subsequently oxidized to acetone (reaction b, Figure 2) [36,37]. In the actinomycete Gordonia sp. strain TY-5, propane is oxidized to isopropanol by a monooxygenase (encoded by prnABC), and then isopropanol is further metabolized to acetone by three distinct NAD+-dependent secondary alcohol dehydrogenases (encoded by adh1, adh2, adh3) . Epoxypropane, formed in certain bacteria by monooxygenation of propene, can undergo isomerization to acetone (reaction c, Figure 2) . Various members of the Vibrio family possess a multistep pathway of leucine catabolism that produces 3-hydroxy-3-methylglutaryl-coenzyme A , which decomposes to yield acetone (reaction d, Figure 2). Finally, other microbes oxidize 2-nitropropane (reaction e, Figure 2), atrazine (reaction f, Figure 2), or other compounds to produce acetone [41,42].
Overview of bacterial acetone metabolism.
In addition to being formed as an intermediate metabolite or product of bacterial metabolic pathways, acetone can serve as a growth-supporting substrate for many diverse bacteria. Previous studies on bacterial acetone metabolism both in vivo and in vitro suggested that acetone can be metabolized in two ways: an oxygen-dependent monooxygenase-catalyzed oxidation to produce acetol (hydroxyacetone), or a CO2-dependent carboxylase-catalyzed carboxylation to produce acetoacetate. Most aerobic bacteria employ the hydroxyacetone pathway, although the corresponding enzyme system is not known [36,37,43,44]. In the propane-utilizing bacterium Gordonia sp. strain TY-5, however, acetone degradation has been shown to be initiated by a Baeyer–Villiger monooxygenase (BVMO) encoded by acmA to produce methyl acetate (reaction g, Figure 2) [38,45]. A hydrolase (encoded by acmB) then splits the ester into acetic acid and methanol (reaction h, Figure 2). BVMOs belong to the class of oxidoreductases and catalyze the insertion of a single oxygen atom between the keto group and the adjacent carbon by a reaction equivalent to a chemical Baeyer–Villiger reaction . BVMOs are flavin (mostly FAD)-dependent and require NAD(P)H to catalyze this reaction. Other types of monooxygenases may be capable of catalyzing terminal hydroxylation of acetone to acetol (reaction i, Figure 2). Acetol cleavage to form acetaldehyde and formaldehyde has been proposed (reaction j, Figure 2) ; however, characterization of an enzymatic activity has not been reported. Although acetol dehydrogenase (reaction k, Figure 2) and methylglyoxal dehydrogenase (reaction l, Figure 2) reactions, which would convert acetol to pyruvate, have been characterized . Finally, an NADPH-dependent acetol monooxygenase catalyzing Baeyer–Villiger chemistry (reaction m, Figure 2) in Mycobacterium sp. has been reported to form hydroxymethylene acetate, which spontaneously decomposes to yield acetic acid and formaldehyde (reaction n, Figure 2) . These BVMOs can be found in a wide range of bacteria where they were also reported to be involved in the catabolism of cycloketones [49–55], aromatic ketones [56,57], or aliphatic open-chain ketones [58,59]. Acetone undergoes CO2-dependent carboxylation, yielding acetoacetate, which is subsequently converted through multiple steps to form two molecules of acetyl-CoA (reaction o, Figure 2) [60,61].
Aerobic propylene and acetone metabolism in
Xanthobacter autotrophicus Py2
Many bacteria have been isolated that are capable of growing with a variety of alkenes and ketones as carbon sources. For example, Xanthobacter sp. [26,62] can utilize propylene, epoxypropane, isopropanol, and acetone as growth substrates. Convergent pathways have been characterized in which both epoxide derivatives of alkenes (epoxypropane) and ketones (acetone) are converted to acetoacetate, which then through the activity of a thiolase results in the formation of acetyl-coenzyme A (acetyl-CoA), a central metabolite. These two convergent pathways involved in the metabolism of these important xenobiotic compounds require, as terminal reactions, unique carboxylations with CO2 as a co-substrate.
The most extensively studied pathway of aliphatic alkene oxidation is that of propylene metabolism in X. autotrophicus Py2 and Rhodococcus rhodochrous B276 [63–69]. The pathway is initiated by an alkene monooxygenase which catalyzes the NADH-dependent conversion of propylene to an enatiomeric mixture of R- and S-epoxypropane. The conversion of both epoxides to a β-ketoacid (acetoacetate) occurs by a three-step process requiring four enzymes (EaCoMT; R-HPCDH; S-HPCDH; 2-KPCC), CO2, NAD+, NADPH, and the small organic cofactor coenzyme M (CoM; 2-mercaptoethanesulfonic acid) [63,70] (Figure 3A). The discovery that CoM is the cofactor for this process was somewhat of a surprise; previously, CoM had only been identified as having a role in methanogenic Archaea where a methyl group is transferred to CoM to form a methyl thioether that is subsequently reduced to methane [71–75]. Interestingly, bacterial CoM is synthesized via a different biosynthetic pathway than in methanogenic archaea, representing a fairly profound case of convergent evolution. The bacterial CoM biosynthetic pathway requires new roles for members of the aspartase/fumarase and pyridoxal phosphate-dependent enzymes superfamilies .
Metabolic pathways of propylene/epoxypropane and isopropanol/acetone in
Epoxyalkane Coenzyme M transferase (EaCoMT) is a zinc-dependent transferase that catalyzes the nucleophilic attack of CoM on short-chain epoxides resulting in ring opening and the formation of hydroxyalkyl-CoM thioether conjugates (Figure 3A). R- and S-hydroxypropyl-CoM dehydrogenases (R- and S-HPCDH) are short-chain dehydrogenases that catalyze the NAD+-dependent oxidation of 2-R- or 2-S-hydroxypropyl CoM (R- and S-HDC) to the achiral product 2-ketopropyl CoM (2-KPC). In this reaction, it was demonstrated through structural characterization and modeling of substrate-bound states of each dehydrogenase that CoM plays a critical role in the appropriate orientation of enantiomers in each enzyme for stereoselective hydride abstraction. The final and most intriguing step of the pathway, from the point of view of mechanistic enzymology, is catalyzed by 2-ketopropyl-CoM oxidoreductase/carboxylase (2-KPCC) (Figure 3A). 2-KPCC is a unique member of the disulfide oxidoreductase (DSOR) enzyme family, which catalyzes the reductive cleavage and carboxylation of 2-KPC to produce acetoacetate and CoM and will be discussed in more detail mechanistically below.
X. autotrophicus Py2 can also utilize either isopropanol or acetone as growth substrates. Isopropanol is converted to acetone by the activity of a secondary alcohol dehydrogenase (Figure 3B). Acetone, either as a growth substrate or produced by the activity of alcohol dehydrogenase, is converted directly to the central metabolite acetoacetate through the activity of a novel ATP-dependent acetone carboxylase (AC) enzyme , and then converted into acetyl-CoA that can be utilized in central metabolic pathways (Figure 3B), as mentioned above.
The 2-ketopropyl-CoM oxidoreductase/carboxylase (2-KPCC) and acetone carboxylase (AC) enzymes
Carboxylases are enzymes that catalyze the incorporation of CO2 into organic substrates. In propylene and acetone metabolism, converting 3-carbon substrates into 4-carbon substrates is required for their assimilation into central metabolism through acetyl-CoA . To form the C–C bond, carboxylation enzymes must catalyze nucleophilic activation of substrates and electrophilic activation of CO2. The final carboxylation step of both propylene and acetone metabolism demonstrates the use of high energy intermediates to overcome the activation energy of bond formation. These intermediates create enolacetone as a strong nucleophile and encapsulate CO2 to prevent side reactions. Structural studies of both 2-KPCC and AC have demonstrated how enolacetone forms and is protected from solvent exposure.
2-KPCC is a homodimeric protein composed of 57 kDa subunits, each containing one molecule of FAD . The deduced amino acid sequence of 2-KPCC indicates that the enzyme is a member of the DSOR family of enzymes [80–82]. Enzymes within this family catalyze diverse reactions involving the two-electron reduction in a substrate. They contain a flavin adenine dinucleotide (FAD) cofactor and a redox-active cysteine pair which participate in the reduction in the substrate molecule. The prototype members of the DSOR family, which have been extensively characterized mechanistically and structurally, are dihydrolipoamide dehydrogenase, glutathione reductase, and mercuric reductase . Members of this family, with the exception of mercuric reductase, cleave a disulfide bond followed by protonation of the leaving group. 2-KPCC, however, contains key differences, which allow it to carry out reductive cleavage and carboxylation at a C–S bond.
Biochemical, mechanistic, and structural studies of 2-KPCC have led to the proposal of a unique catalytic cycle for the enzyme [79,83–85]. The reaction scheme (Figure 4A) and the solved structure of 2-KPCC (Figure 4B) are shown in Figure 4. Like DSORs, the catalytic cycle is initiated when NADPH reduces the enzyme FAD, which then reduces the oxidized cysteine pair (Cys82 and Cys87 in the primary sequence of 2-KPCC). The substrate 2-KPC then binds, followed by a nucleophilic attack of the interchange cysteine thiol on the substrate thioether sulfur, resulting in heterolytic cleavage of the S–C bond. This step represents a fundamentally novel reaction with respect to the DSOR family, in that the cysteine attacks a thioether rather than a disulfide, as in all members of the family except mercuric reductase. The cleavage of the thioether results in the formation of a stabilized carbanion of acetone and a mixed disulfide of cysteine and CoM, both of which are unique to the mechanism of 2-KPCC. Enolacetone serves as the nucleophile for an attack on CO2 in the carboxylation step, forming acetoacetate. In the absence of CO2, enolacetone will be protonated to form side product acetone. The catalytic cycle is completed by reduction in the mixed disulfide of CoM and the interchange thiol, forming free CoM and the mixed disulfide cysteine pair.
Enzymatic reaction and structure of 2-KPCC.
Structural characterization of 2-KPCC with and without substrate has also provided further insight into how 2-KPCC directs carboxylation [79,84,86]. The structure of the substrate-bound state clearly indicates that the substrate binds the active site through the strong electrostatic interactions of two arginine side chains with the sulfonate group of the substrate. This provides the proper alignment of the substrate with the redox-active disulfide for facile cleavage. Furthermore, a histidine-oriented water molecule could interact with the substrate keto group to stabilize the enolate intermediate . A comparison of the structures in the native and substrate-bound states indicated that 2-KPC induces a conformational change that results in the collapse of the substrate access channel. A cis-proline flanked loop region, which is stabilized by a divalent metal ion, acts as a shield for the substrate . This encapsulation of the substrate is unique to 2-KPCC, whereas the DSOR members maintain an open structure, permitting substrate access for protonation.
2-KPCC also promotes the unusual carboxylation of a C–S bond. DSORs contain a conserved HXXXXE catalytic dyad; however, 2-KPCC contains a FXXXXH dyad (Figure 4C). It has been proposed that this modification excludes an acid–base from the active site to aid in promoting carboxylation in 2-KPCC [87,88]. Mutagenesis of 2-KPCC's active site dyad to mimic that of DSORs shifted the product formation to favor protonation of the substrate to acetone, suggesting the specificity of this residue in the active site (native phenylalanine (Phe) vs histidine (His)) indeed plays a role in product identity . In addition to providing a source of protons in DSORs, the His lowers the pKa of the CysINT such that it can reduce its substrate. With the replacement with Phe in 2-KPCC, its CysINT pKa is not lowered but rather CysINT and CysCT share a proton, protecting the enolacetone intermediate from free protons and rendering the CysCT an internal acid–base (Figure 4A) . Such proton sharing between two cysteines stabilize a reactive thiolate anion as observed in thioredoxin . In thioredoxin, the reactive thiolate also resides in a hydrophobic environment, much like 2-KPCC. Proton sharing between the reactive cysteines in 2-KPCC, thereby, is thought to stabilize the cysteine (CysINT) which interacts with substrate, enabling the splitting of the strong C–S bond.
Acetone carboxylase (AC)
Acetone metabolism has been biochemically characterized from multiple proteobacteria classes, with the most extensive focus on the α-proteobacteria X. autotrophicus Py2 and β-proteobacteria Aromatoleum aromaticum. While the majority of the biochemical and structural work has been done in X. autotrophicus Py2, reports from A. aromaticum show fundamental differences in the reported reaction stoichiometry and metal content. A. aromaticum AC was described as hydrolyzing two ATP to two AMP and four inorganic phosphates (Pi) for each molecule of acetone carboxylated . This is in contrast with AC from X. autotrophicus Py2, which only requires a single molecule of ATP hydrolyzed to AMP and two Pi per acetone carboxylated . AC from A. aromaticum has been reported to contain Fe at its active site instead of Mn2+ present in the AC from X. autotrophicus Py2 [60,68,90].
While the active site of AC has not been characterized to the extent of 2-KPCC, structural studies have allowed a mechanism to be proposed for X. autotrophicus Py2 AC . AC has two heterotrimeric αβγ subunits joined by the interacting α-subunits to form a dimeric core. The α-subunits contain a catalytic Mn2+ site while the β-subunits contain the nucleotide-binding site (Figure 5A). The structure of AC was determined in multiple conformational states, demonstrating significant conformational changes that occur during catalytic turnover . The structure shows the nucleotide and Mn2+ active sites located more than 40 Å away from each other. The distance between the active sites can be rationalized by comparing the conformational states. The changes in these states show that nucleotide-binding communicates with the Mn2+ active site through an extensive 11–18 Å rotation of the β subunit (Figure 5B). It has been proposed that the rotation encapsulates the substrates ATP, , and acetone at the nucleotide-binding site. Acetone is phosphorylated through a concerted reaction involving the abstraction of an α-proton with the hydrolysis of the γ-phosphoryl group of ATP to the carbonyl oxygen of acetone, generating the phosphoenolacetone intermediate. The is proposed to be phosphorylated by the β-phosphoryl group of ATP, creating AMP and carboxyphosphate (Figure 5C). These reactive intermediates are protected from solvent hydrolysis by the substrate tunnel and can freely diffuse inside the enzyme. During the conformational shift upon AMP binding, α-Glu89 co-ordinates to the Mn2+ active site, causing the proximal helix to become disordered. This disordering of the proximal helix allows for access of the phosphorylated intermediates to the Mn2+ active site. The intermediates will then co-ordinate with the Mn2+ and displace α-Glu89, subsequently closing off access to the Mn2+ active site. The coordination of the phosphorylated intermediates has been proposed to facilitate two reaction steps: (1) carboxyphosphate decay to yield electrophilic CO2 and (2) the creation of enolacetone through the dephosphorylation of phosphoenolacetone (Figure 5C). This in situ creation of enolacetone and CO2, which combine to form acetoacetate, is similar to the proposed the 2-KPCC-catalyzed reaction. The formation of the product and release of inorganic phosphates allows the enzyme to reset. The conformational states and proposed mechanism show a cyclic nature to the activity of the enzyme, where substrate binding causes rotation of the β-subunit, encapsulating the reactive intermediates. The rotation opens access to the Mn2+ active site where the intermediates can diffuse to co-ordinate with the Mn2+. Once the intermediates displace α-Glu89, the tunnel closes and the cycle can start again.
Structure and mechanism of acetone carboxylase (AC).
Studies on propylene and acetone metabolism in bacteria have identified a central role for CO2 and specifically CO2 fixation reactions in these processes. The CO2 fixing enzymes of the pathways, 2-KPCC and AC, are distinct carboxylases with unique molecular properties and cofactor requirements. Characterization of these novel metabolic pathways coupled with advancement in metabolic engineering of microorganisms can provide a blueprint to promote coupling the metabolism of potentially deleterious compounds to CO2 fixation.
The authors declare that there are no competing interests associated with the manuscript.
This work was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award DE-FG02×04ER15563 to J.W.P., J.L.D., and B.B. Partial salary support for J.W.P. was supported by the USDA National Institute of Food and Agriculture, Hatch umbrella project #1015621.