The 3-hydroxyacyl-CoA epimerase activity of rat liver peroxisomes is due to the combined actions of two enoyl-CoA hydratases: a revision of the epimerase-dependent pathway of unsaturated fatty acid oxidation

Chromatography of a rat liver extract on DEAE-cellulose resulted in the near total loss of 3-hydroxyacyl-CoA epimerase activity. The activity was regained either when fractions were recombined or when purified crotonase was added to the early column fractions. A new enoyl-CoA hydratase present in these early fractions catalyzes the conversion of D-3-hydroxyacyl-CoA to 2-trans-enoyl-CoA which can be hydrated by crotonase or the peroxisomal bifunctional enzyme to L-3-hydroxyacyl-CoA. Thus, the 3-hydroxyacyl-CoA epimerase activity is due to the combined actions of two enoyl-CoA hydratases with opposite stereospecificities.     

Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities

Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.     

The 1.3 A crystal structure of human mitochondrial Delta3-Delta2-enoyl-CoA isomerase shows a novel mode of binding for the fatty acyl group

The crystal structure of Delta3-Delta2-enoyl-CoA isomerase from human mitochondria (hmEci), complexed with the substrate analogue octanoyl-CoA, has been refined at 1.3 A resolution. This enzyme takes part in the beta-oxidation of unsaturated fatty acids by converting both cis-3 and trans-3-enoyl-CoA esters (with variable length of the acyl group) to trans-2-enoyl-CoA. hmEci belongs to the hydratase/isomerase (crotonase) superfamily. Most of the enzymes belonging to this superfamily are hexamers, but hmEci is shown to be a trimer. The mode of binding of the ligand, octanoyl-CoA, shows that the omega-end of the acyl group binds in a hydrophobic tunnel formed by residues of the loop preceding helix H4 as well as by side-chains of the kinked helix H9. From the structure of the complex it can be seen that Glu136 is the only catalytic residue. The importance of Glu136 for catalysis is confirmed by mutagenesis studies. A cavity analysis shows the presence of two large, adjacent empty hydrophobic cavities near the active site, which are shaped by side-chains of helices H1, H2, H3 and H4. The structure comparison of hmEci with structures of other superfamily members, in particular of rat mitochondrial hydratase (crotonase) and yeast peroxisomal enoyl-CoA isomerase, highlights the variable mode of binding of the fatty acid moiety in this superfamily.     

Mutation of Lys242 allows Delta3-Delta2-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Delta3-Delta2-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k(cat) value of 1.0 s(-1) for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.     

Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1

Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.     

Different chain length specificities of peroxisomal and mitochondrial enoyl-CoA hydratases

Peroxisomes and mitochondria were purified from rat liver by differential and equilibrium density centrifugation. Enoyl-CoA hydratase activity was assayed with two substrates: crotonyl-CoA (C4) and dodecenoyl-CoA (C12). The chain length specificity of the hydratase(s) in the two organelles differed strikingly: the ratio of activity on the C4 substrate/C12 substrate was 8–10 for the peak mitochondrial fraction and about 2 for the peak peroxisomal fraction. The subcellular distribution of the hydratase activity also depended on chain length. Peroxisomes contain 30–50% of the dodecenoyl-CoA hydratase activity but only 9–15% of the crotonase activity of rat liver.     

Mutation of Lys242 allows Δ-Δ-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Δ³-Δ²-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k_cat value of 1.0 s^− 1 for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.     

Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity

Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.     

Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template.

The structures and chemical pathways associated with the members of the 2-enoyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a common active site design provides the members of this family with a CoA binding site, an expandable acyl binding pocket, an oxyanion hole for binding/polarizing the thioester C=O, and multiple active site stations for the positioning of acidic and basic amino acid side chains for use in proton shuttling. It is hypothesized that this active site template can be tailored to catalyze a wide range of chemical transformations through strategic positioning of acid/base residues among the active site stations. To test this hypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isomerase family, 4-chlorobenzoyl-CoA dehalogenase, was altered by site-directed mutagenesis to include the two glutamate residues functioning in acid/base catalysis in a second family member, crotonase. Catalysis of the syn hydration of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogenase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA dehalogenase at kcat = 0.06 s-1 and Km = 50 microM.     

A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2

2-Enoyl-CoA hydratase 2, a part from multifunctional enzyme type 2, hydrates trans-2-enoyl-CoA to 3-hydroxyacyl-CoA in the (3R)-hydroxy-dependent route of peroxisomal beta-oxidation of fatty acids. Unliganded and (3R)-hydroxydecanoyl coenzyme A-complexed crystal structures of 2-enoyl-CoA hydratase 2 from Candida tropicalis multifunctional enzyme type 2 were solved to 1.95- and 2.35-A resolution, respectively. 2-Enoyl-CoA hydratase 2 is a dimeric, alpha+beta protein with a novel quaternary structure. The overall structure of the two-domain subunit of eukaryotic 2-enoyl-CoA hydratase 2 resembles the homodimeric, hot dog fold structures of prokaryotic (R)-specific 2-enoyl-CoA hydratase and beta-hydroxydecanoyl thiol ester dehydrase. Importantly, though, the eukaryotic hydratase 2 has a complete hot dog fold only in its C-domain, whereas the N-domain lacks a long central alpha-helix, thus creating space for bulkier substrates in the binding pocket and explaining the observed difference in substrate preference between eukaryotic and prokaryotic enzymes. Although the N- and C-domains have an identity of <10% at the amino acid level, they share a 50% identity at the nucleotide level and fold similarly. We suggest that a subunit of 2-enoyl-CoA hydratase 2 has evolved via a gene duplication with the concomitant loss of one catalytic site. The hydrogen bonding network of the active site of 2-enoyl-CoA hydratase 2 resembles the active site geometry of mitochondrial (S)-specific 2-enoyl-CoA hydratase 1, although in a mirror image fashion. This arrangement allows the reaction to occur by similar mechanism, supported by mutagenesis and mechanistic studies, although via reciprocal stereochemistry.     

Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes

Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. Two alternative pathways have been described to degrade these fatty acids. One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases. Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway. We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle. Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.     

Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product.

The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain. The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids. The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000). Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex. The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S. cerevisiae and the gene product purified to apparent homogeneity. A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified. Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA). The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity. These results clearly demonstrate that the beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate.     

Involvement of the fatty acid oxidation complex in acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli

The activity of acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli was enhanced when the organism was grown on oleic acid as the sole carbon source, but not detected when grown on glucose. Antibodies raised against fatty acid oxidation complex of E. coli inhibited both the reaction catalyzed by crotonase and the chain elongation in a similar manner, showing that the oxidation complex participates in the chain elongation. The activities of condensation and the activities of NADH- and NADPH-dependent 3-ketoacyl reduction in the cell-free extract were precipitated by antibodies to the complex in parallel with those of 3-ketoacyl-CoA thiolase and crotonase. These results together with the presence of NADPH-dependent trans-2-enoyl-CoA reductase in E. coli (Mizugaki, et al. (1982) Chem. Pharm. Bull. 30, 2503-2511) indicate that the acetyl-CoA-dependent chain elongation of fatty acids in E. coli occurs by the reversal of fatty acid oxidation other than the step of enoyl reduction.     

Orientation of coenzyme A substrates, nicotinamide and active site functional groups in (Di)enoyl-coenzyme A reductases

The stereochemical course of reduction of dienoyl-coenzyme A (CoA) thiolesters catalyzed by the 2,4-dienoyl-CoA reductase from rat liver mitochondria was investigated. The configuration of the double bond in the 3-enoyl-CoA products was determined by (1)H NMR, and experiments to determine the stereochemical course of reduction at Calpha and Cdelta by use of 4-(2)H-labeled beta-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), were conducted in H(2)O and D(2)O. Defining the diastereoselectivity of the reaction, catalyzed by the Delta(3),Delta(2)-enoyl-CoA isomerase, facilitated the determination of the stereochemical course of reduction by 2, 4-dienoyl-CoA reductase. The absence of solvent exchange of the proton transferred during the Delta(3),Delta(2)-enoyl-CoA isomerase catalyzed equilibration of trans-2- and trans-3-enoyl-CoAs, coupled with the strong sequence homology to enoyl-CoA hydratase support the intramolecular suprafacial transfer of the pro-2R proton of trans-3-enoyl-CoA to the pro-4R position of trans-2-enoyl-CoA. The results indicate that the configuration of the double bond of the 3-enoyl-CoA product is trans and that a general acid-catalyzed addition of a solvent derived proton/deuteron occurs on the si face at Calpha of the dienoyl-CoA. The addition of the pro-4S hydrogen from NADPH occurs on the si face at Cdelta of trans-2, cis-4-dienoyl-CoA and on the re face at Cdelta of trans-2, trans-4-dienoyl-CoA. The stereochemical course of reduction of InhA, an enoyl-thiolester reductase from Mycobacterium tuberculosis, was also determined by use of ?4-(2)HNADH in D(2)O. The reduction of trans-2-octenoyl-CoA catalyzed by InhA resulted in the syn addition of (2)H(2) across the double bond yielding (2R,3S)-?2, 3-(2)H(2)?ctanoyl-CoA. In the crystal structure of the InhA ternary complex, the residue donating the proton to Calpha could not be identified ?Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittman, R., and Sacchettini, J. C. (1999) J. Biol. Chem. 274, 15582-15589. The current results place further restrictions on the source of the proton and suggest the reduction is stepwise.     

Evolution of function in the crotonase superfamily: (3S)-methylglutaconyl-CoA hydratase from Pseudomonas putida

Members of the enoyl-CoA hydratase (crotonase) superfamily catalyze different overall reactions that utilize a common catalytic strategy delivered by a shared structural scaffold; the substrates are usually acyl esters of coenzyme A, and the intermediates are usually thioester enolate anions stabilized by a conserved oxyanion hole. In many bacterial genomes, orthologous members that contain homologues of acid/base catalyst Glu164 but not of Glu144 in rat mitochondrial crotonase are encoded by operons of which the functions have not been assigned. Focusing on the orthologues from Pseudomonas aeruginosa and P. putida, we have determined that these operons encode enzymes in leucine catabolism with the unknown enzyme assigned as (3S)-methylglutaconyl-CoA hydratase (MGCH), which catalyzes the syn-hydration of (E)-3-methylglutaconyl-CoA to (3S)-hydroxymethylglutaryl-CoA. The discovery that bacterial MGCHs catalyze hydration of enoyl-CoAs utilizing a single active-site residue contrasts with the paradigm crotonases as well as with the recently identified mammalian MGCHs that use homologues of both Glu144 and Glu164 in crotonase. Substrate analogues lacking a gamma-carboxylate have been shown to be competitive inhibitors of the enzyme, and installation of a glutamate for the "missing" homologue of Glu144 fails to introduce hydratase activity with the substrate analogues. Thus, bacterial MGCHs may provide an example of opportunistic evolution in which a carboxylate group of the substrate functionally replaces one of the active site glutamate residues in the reactions catalyzed by crotonases and the eukaryotic MGCHs.     

Inhibition of enoyl-CoA hydratase by long-chain L-3-hydroxyacyl-CoA and its possible effect on fatty acid oxidation.

The kinetics of bovine liver enoyl-CoA hydratase (EC 4.2.1.17) or crotonase with 2-trans-hexadecenoyl-CoA as a substrate were studied because different rates were obtained with two assay methods based on measurements of substrate utilization and product formation, respectively. L-3-Hydroxyhexadecanoyl-CoA, the product of the crotonase-catalyzed hydration of 2-trans-hexadecenoyl-CoA, was found to be a strong competitive inhibitor of the enzyme with a Ki of 0.35 microM. In contrast the short-chain product, L-3-hydroxybutyryl-CoA, is a weak competitive inhibitor with a Ki of 37 microM. L-3-Hydroxyhexadecanoyl-CoA is a much stronger inhibitor of crotonase than are other short-chain and long-chain intermediates of beta-oxidation and crotonase is more severely inhibited by this compound than are all beta-oxidation enzymes tested so far. Determination of true kinetic parameters for the crotonase-catalyzed hydration of long-chain substrates requires the removal of product in a coupled assay. When this was done, the Km for 2-trans-hexadecenoyl-CoA with bovine liver crotonase was found to be only 9 microM. It is suggested that under conditions of restricted beta-oxidation, when 3-hydroxyacyl-CoAs accumulate in mitochondria, the inhibition of crotonase by long-chain 3-hydroxyacyl-CoAs may limit the further degradation of medium-chain and short-chain intermediates of beta-oxidation.     

Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.     

Solubilization and Purification of Rat Liver Microsomal Trans-2-Enoyl-CoA Hydratase

It is well known that fatty acid chain elongation involves four steps : (a) condensation of a primer with malonyl-CoA to form the β-keto acyl CoA; (b) reduction of the resulting β -keto acyl CoA to a secondary alcohol, β-hydroxyl acyl CoA; (c) dehydration of the alcohol to form trans-2-enoyl CoA; and (d) reduction of the trans-2-enoyl CoA to give an acid 2 carbon atoms longer than the primer. ^1–2The enzyme involved in step (c) is trans-2-enoyl-CoA hydratase, It can perform a reversible hydration of α, β-unsaturated fatty acid chain elongation. ³A partial purification of this enzyme has been reported by Bernert and Sprecher. ⁴The purification of enoyl-CoA hydratase from mycobacterium smegmatis ⁵and from rat mitochondria have also been reported; ⁶however, the purification of enoyl-hydratase from rat liver has not been reported.     

Crystal Structure of Liganded Rat Peroxisomal Multifunctional Enzyme Type 1: A FLEXIBLE MOLECULE WITH TWO INTERCONNECTED ACTIVE SITES*

The crystal structure of the full-length rat peroxisomal multifunctional enzyme, type 1 (rpMFE1), has been determined at 2.8 Å resolution. This enzyme has three catalytic activities and two active sites. The N-terminal part has the crotonase fold, which builds the active site for the Δ³,Δ²-enoyl-CoA isomerase and the Δ²-enoyl-CoA hydratase-1 catalytic activities, and the C-terminal part has the (3S)-hydroxyacyl-CoA dehydrogenase fold and makes the (3S)-hydroxyacyl-CoA dehydrogenase active site. rpMFE1 is a multidomain protein having five domains (A–E). The crystal structure of full-length rpMFE1 shows a flexible arrangement of the A-domain with respect to the B–E-domains. Because of a hinge region near the end of the A-domain, two different positions of the A-domain were observed for the two protein molecules (A and B) of the asymmetric unit. In the most closed conformation, the mode of binding of CoA is stabilized by domains A and B (helix-10), as seen in other crotonase fold members. Domain B, although functionally belonging to the N-terminal part, is found tightly associated with the C-terminal part, i.e. fixed to the E-domain. The two active sites of rpMFE1 are ∼40 Å apart, separated by a tunnel, characterized by an excess of positively charged side chains. Comparison of the structures of rpMFE1 with the monofunctional crotonase and (3S)-hydroxyacyl-CoA dehydrogenase superfamily enzymes, as well as with the bacterial α₂β₂-fatty acid oxidation multienzyme complex, reveals that this tunnel could be important for substrate channeling, as observed earlier on the basis of the kinetics of rpMFE1 purified from rat liver.     

Carboxymethylproline synthase (CarB), an unusual carbon-carbon bond-forming enzyme of the crotonase superfamily involved in carbapenem biosynthesis

Carboxymethylproline synthase (CarB) catalyzes the committed step in the biosynthesis of (R)-1-carbapen-2-em-3-carboxylate, the simplest member of the carbapenem family of beta-lactam antibiotics, some of which are used clinically. CarB displays sequence homology with members of the crotonase family including enoyl-CoA hydratase (crotonase) and methylmalonyl-CoA decarboxylase. The CarB reaction has been proposed to comprise condensation of acetyl coenzyme A (AcCoA) and glutamate semi-aldehyde to give (2S,5S)-carboxymethylproline ((2S,5S)-CMP). (2S,5S)-CMP is then cyclized in an ATP-driven reaction catalyzed by CarA to give a carbapenam, which is subsequently epimerized and desaturated to give a carbapenem in a CarC-mediated reaction. Here we report the purification of recombinant CarB and that it exists predominantly in a trimeric form as do other members of the crotonase family. AcCoA was not found to be a substrate for CarB. Instead malonyl-CoA was found to be a substrate, efficiently producing (2S,5S)-CMP in the presence of glutamate semi-aldehyde. In the absence of glutamate semi-aldehyde, mass spectrometric analysis indicated that CarB catalyzed the decarboxylation of malonyl-CoA to AcCoA. The reactions of CarB, CarA, and CarC were coupled in vitro demonstrating the viability of malonyl-CoA as a carbapenem precursor. CarB was also shown to accept methylmalonyl CoA as a substrate to form 6-methyl-(2S,5S)CMP, which in turn is a substrate for CarA. The implications of the results for the biosynthesis of both carbapenem-3-carboxylate and C-2/C-6-substituted carbapenems, such as thienamycin, are discussed.