Studies on the peroxisomal multifunctional enzyme type-1:domain structure with special reference to the hydratase/isomerase fold

Abstract The peroxisomal multifunctional enzyme type-1 (perMFE-1) is a monomeric protein of β-oxidation possessing 2-enoyl-CoA hydratase-1, Δ3-Δ 2-enoyl-CoA isomerase, and (3S)-hydroxyacyl-CoA dehydrogenase activities. The amino-terminal part of perMFE-1 shows sequence similarity to mitochondrial 2-enoyl-CoA hydratases (ECH-1) and Δ3-Δ 2-enoyl-CoA isomerases, and belongs to the hydratase/isomerase superfamily. Family members with known structures are either homotrimers or homohexamers. The purpose of this work was to elucidate the structure-function relationship of the rat perMFE-1 with special reference to the hydratase/isomerase fold. The structural adaptations required for binding of a long chain fatty acyl-CoA were studied with rat ECH-1 via co-crystallization with octanoyl-CoA. The crystal structure revealed that the long chain fatty acyl-CoA is bound in an extended conformation. This is possible because, a flexible loop moves aside and opens a tunnel, which traverses the subunit from the solvent space to the intertrimer space. Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by ECH-1. In the present work the enzymological properties of Glu144Ala and Glu164Ala variants of ECH-1 were studied. The catalytic activity of hydration was reduced about 2000-fold. It was also demonstrated that rat ECH-1 is capable of catalyzing isomerization. The replacement of Glu164 with alanine reduced the isomerase activity 1000-fold, confirming the role of Glu164 in both the hydratase and isomerase reactions. The structural factors favoring the hydratase over the isomerase reaction were addressed studying the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. These mutants had similar enzymatic properties to wild type, thus the catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on interaction with the Gln162 side chain. The perMFE-1 was divided into five functional domains based on amino acid sequence comparisons with the homologous proteins with known structures. Deletion variants of perMFE-1 showed that the folding of an enzymatically active amino-terminal hydratase/isomerase domain requires stabilizing interactions from the two carboxy-terminal domains of perMFE-1. The last carboxy-terminal domain is also required for the folding of the dehydrogenase part of perMFE-1. The dehydrogenase part of perMFE-1 was crystallized

Structural characterization of a mitochondrial 3-ketoacyl-CoA (T1)-like thiolase from Mycobacterium smegmatis

Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) from Mycobacterium smegmatis in apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domain via flexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The L alpha 1-covering loop-L alpha 2 region, together with the N beta 2-N alpha 2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains

Structures of lactaldehyde reductase, FucO, link enzyme activity to hydrogen bond networks and conformational dynamics

A group-III iron containing 1,2-propanediol oxidoreductase, FucO, (also known as lactaldehyde reductase) from Escherichia coli was examined regarding its structure–dynamics–function relationships in the catalysis of the NADH-dependent reduction of (2S)-lactaldehyde. Crystal structures of FucO variants in the presence or absence of cofactors have been determined, illustrating large domain movements between the apo and holo enzyme structures. Different structures of FucO variants co-crystallized with NAD+ or NADH together with substrate further suggest dynamic properties of the nicotinamide moiety of the coenzyme that are important for the reaction mechanism. Modelling of the native substrate (2S)-lactaldehyde into the active site can explain the stereoselectivity exhibited by the enzyme, with a critical hydrogen bond interaction between the (2S)-hydroxyl and the side-chain of N151, as well as the previously experimentally demonstrated pro-(R) selectivity in hydride transfer from NADH to the aldehydic carbon. Furthermore, the deuterium kinetic isotope effect of hydride transfer suggests that reduction chemistry is the main rate-limiting step for turnover which is not the case in FucO catalysed alcohol oxidation. We further propose that a water molecule in the active site – hydrogen bonded to a conserved histidine (H267) and the 2′-hydroxyl of the coenzyme ribose – functions as a catalytic proton donor in the protonation of the product alcohol. A hydrogen bond network of water molecules and the side-chains of amino acid residues D360 and H267 links bulk solvent to this proposed catalytic water molecule

Organization of the multifunctional enzyme type 1: interaction between N- and C-terminal domains is required for the hydratase-1/isomerase activity.

Rat peroxisomal multifunctional enzyme type 1 (perMFE-1) is a monomeric protein of beta-oxidation. We have defined five functional domains (A, B, C, D and E) in the perMFE-1 based on comparison of the amino acid sequence with homologous proteins from databases and structural data of the hydratase-1/isomerases (H1/I) and (3 S )-hydroxyacyl-CoA dehydrogenases (HAD). Domain A (residues 1-190) comprises the H1/I fold and catalyses both 2-enoyl-CoA hydratase-1 and Delta(3)-Delta(2)-enoyl-CoA isomerase reactions. Domain B (residues 191-280) links domain A to the (3 S )-dehydrogenase region, which includes both domain C (residues 281-474) and domain D (residues 480-583). Domains C and D carry features of the dinucleotide-binding and the dimerization domains of monofunctional HADs respectively. Domain E (residues 584-722) has sequence similarity to domain D of the perMFE-1, which suggests that it has evolved via partial gene duplication. Experiments with engineered perMFE-1 variants demonstrate that the H1/I competence of domain A requires stabilizing interactions with domains D and E. The variant His-perMFE (residues 288-479)Delta, in which the domain C is deleted, is stable and has hydratase-1 activity. It is proposed that the extreme C-terminal domain E in perMFE-1 serves the following three functions: (i) participation in the folding of the N-terminus into a functionally competent H1/I fold, (ii) stabilization of the dehydrogenation domains by interaction with the domain D and (iii) the targeting of the perMFE-1 to peroxisomes via its C-terminal tripeptide

Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1

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Thiolase:a versatile biocatalyst employing coenzyme A–thioester chemistry for making and breaking C–C bonds

Abstract Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: (a) a nucleophilic cysteine, which forms a covalent intermediate, and (b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed

Crystal structures and kinetic studies of a laboratory evolved aldehyde reductase explain the dramatic shift of its new substrate specificity

The Fe2+-dependent E. coli enzyme FucO catalyzes the reversible interconversion of short-chain (S)-lactaldehyde and (S)-1,2-propanediol, using NADH and NAD+ as cofactors, respectively. Laboratory-directed evolution experiments have been carried out previously using phenylacetaldehyde as the substrate for screening catalytic activity with bulky substrates, which are very poorly reduced by wild-type FucO. These experiments identified the N151G/L259V double mutant (dubbed DA1472) as the most active variant with this substrate via a two-step evolutionary pathway, in which each step consisted of one point mutation. Here the crystal structures of DA1472 and its parent D93 (L259V) are reported, showing that these amino acid substitutions provide more space in the active site, though they do not cause changes in the main-chain conformation. The catalytic activity of DA1472 with the physiological substrate (S)-lactaldehyde and a series of substituted phenylacetaldehyde derivatives were systematically quantified and compared with that of wild-type as well as with the corresponding point-mutation variants (N151G and L259V). There is a 9000-fold increase in activity, when expressed as kcat/KM values, for DA1472 compared with wild-type FucO for the phenylacetaldehyde substrate. The crystal structure of DA1472 complexed with a non-reactive analog of this substrate (3,4-dimethoxyphenylacetamide) suggests the mode of binding of the bulky group of the new substrate. These combined structure–function studies therefore explain the dramatic increase in catalytic activity of the DA1472 variant for bulky aldehyde substrates. The structure comparisons also suggest why the active site in which Fe2+ is replaced by Zn2+ is not able to support catalysis