Correction:How the COVID-19 pandemic highlights the necessity of animal research (vol 30, pg R1014, 2020)

(Current Biology 30, R1014–R1018; September 21, 2020) As a result of an author oversight in the originally published version of this article, a number of errors were introduced in the author list and affiliations. First, the middle initials were omitted from the names of several authors. Second, the surname of Dr. van Dam was mistakenly written as “Dam.” Third, the first name of author Bernhard Englitz was misspelled as “Bernard” and the surname of author B.J.A. Pollux was misspelled as “Pullox.” Finally, Dr. Keijer's first name was abbreviated rather than written in full. These errors, as well as various errors in the author affiliations, have now been corrected online

Enzymes of the crotonase superfamily:diverse assembly and diverse function

Abstract The crotonase fold is generated by a framework of four repeats of a ββα-unit, extended by two helical regions. The active site of crotonase superfamily (CS) enzymes is located at the N-terminal end of the helix of the third repeat, typically being covered by a C-terminal helix. A major subset of CS-enzymes catalyzes acyl-CoA-dependent reactions, allowing for a diverse range of acyl-tail modifications. Most of these enzymes occur as trimers or hexamers (dimers of trimers), but monomeric forms are also observed. A common feature of the active sites of CS-enzymes is an oxyanion hole, formed by two peptide-NH hydrogen bond donors, which stabilises the negatively charged thioester oxygen atom of the reaction intermediate. Structural properties and possible use of these enzymes for biotechnological applications are discussed

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

Structural Enzymological studies of 2-Enoyl-Thioester Reductase of the Human Mitochondrial FAS II pathway: New Insights into Its Substrate Recognition Properties

Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway

Insights into the stability and substrate specificity of the E. coli aerobic β-oxidation trifunctional enzyme complex

Abstract Degradation of fatty acids by the β-oxidation pathway results in the formation of acetyl-CoA which enters the TCA cycle for the production of ATP. In E. coli, the last three steps of the β-oxidation are catalyzed by two heterotetrameric α₂β₂ enzymes namely the aerobic trifunctional enzyme (EcTFE) and the anaerobic TFE (anEcTFE). The α-subunit of TFE has 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities whereas the β-subunit is a thiolase with 3-ketoacyl-CoA thiolase (KAT) activity. Recently, it has been shown that the two TFEs have complementary substrate specificities allowing for the complete degradation of long chain fatty acyl-CoAs into acetyl-CoA under aerobic conditions. Also, it has been shown that the tetrameric EcTFE and anEcTFE assemblies are similar to the TFEs of Pseudomans fragi and human, respectively. Here the properties of the EcTFE subunits are further characterized. Strikingly, it is observed that when expressed separately, EcTFE-α is a catalytically active monomer whereas EcTFE-β is inactive. However, when mixed together active EcTFE tetramer is reconstituted. The crystal structure of the EcTFE-α chain is also reported, complexed with ATP, bound in its HAD active site. Structural comparisons show that the EcTFE hydratase active site has a relatively small fatty acyl tail binding pocket when compared to other TFEs in good agreement with its preferred specificity for short chain 2E-enoyl-CoA substrates. Furthermore, it is observed that millimolar concentrations of ATP destabilize the EcTFE complex, and this may have implications for the ATP-mediated regulation of β-oxidation in E. coli

Crystallographic binding studies of rat peroxisomal multifunctional enzyme, type-1 (MFE1) with 3-ketodecanoyl-CoA:capturing active and inactive states of its hydratase and dehydrogenase catalytic sites

Abstract The peroxisomal multifunctional enzyme type 1 (MFE1) catalyzes two successive reactions in the β-oxidation cycle: the 2E-enoyl-CoA hydratase (ECH) and NAD⁺-dependent 3S-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 Å resolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD⁺ bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part

Structural basis for different membrane-binding properties of E. coli anaerobic and human mitochondrial β-oxidation trifunctional enzymes

Abstract Facultative anaerobic bacteria such as Escherichia coli have two α₂β₂ heterotetrameric trifunctional enzymes (TFE), catalyzing the last three steps of the β-oxidation cycle: soluble aerobic TFE (EcTFE) and membrane-associated anaerobic TFE (anEcTFE), closely related to the human mitochondrial TFE (HsTFE). The cryo-EM structure of anEcTFE and crystal structures of anEcTFE-α show that the overall assembly of anEcTFE and HsTFE is similar. However, their membrane-binding properties differ considerably. The shorter A5-H7 and H8 regions of anEcTFE-α result in weaker α-β as well as α-membrane interactions, respectively. The protruding H-H region of anEcTFE-β is therefore more critical for membrane-association. Mutational studies also show that this region is important for the stability of the anEcTFE-β dimer and anEcTFE heterotetramer. The fatty acyl tail binding tunnel of the anEcTFE-α hydratase domain, as in HsTFE-α, is wider than in EcTFE-α, accommodating longer fatty acyl tails, in good agreement with their respective substrate specificities

Phylogenetic relationships and classification of thiolases and thiolase-like proteins of Mycobacterium tuberculosis and Mycobacterium smegmatis

Thiolases are enzymes involved in lipid metabolism. Thiolases remove the acetyl-CoA moiety from 3-ketoacyl-CoAs in the degradative reaction. They can also catalyze the reverse Claisen condensation reaction, which is the first step of biosynthetic processes such as the biosynthesis of sterols and ketone bodies. In human, six distinct thiolases have been identified. Each of these thiolases is different from the other with respect to sequence, oligomeric state, substrate specificity and subcellular localization. Four sequence fingerprints, identifying catalytic loops of thiolases, have been described. In this study genome searches of two mycobacterial species (Mycobacterium tuberculosis and Mycobacterium smegmatis), were carried out, using the six human thiolase sequences as queries. Eight and thirteen different thiolase sequences were identified in M. tuberculosis and M. smegmatis, respectively. In addition, thiolase-like proteins (one encoded in the Mtb and two in the Msm genome) were found. The purpose of this study is to classify these mostly uncharacterized thiolases and thiolase-like proteins. Several other sequences obtained by searches of genome databases of bacteria, mammals and the parasitic protist family of the Trypanosomatidae were included in the analysis. Thiolase-like proteins were also found in the trypanosomatid genomes, but not in those of mammals. In order to study the phylogenetic relationships at a high confidence level, additional thiolase sequences were included such that a total of 130 thiolases and thiolase-like protein sequences were used for the multiple sequence alignment. The resulting phylogenetic tree identifies 12 classes of sequences, each possessing a characteristic set of sequence fingerprints for the catalytic loops. From this analysis it is now possible to assign the mycobacterial thiolases to corresponding homologues in other kingdoms of life. The results of this bioinformatics analysis also show interesting differences between the distributions of M. tuberculosis and M. smegmatis thiolases over the 12 different classes. (C) 2014 Elsevier Ltd. All rights reserved

Substrate specificity and conformational flexibility properties of the Mycobacterium tuberculosis β-oxidation trifunctional enzyme

Abstract The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α₂β₂ tetrameric enzyme. The α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) activity. Enzyme kinetic data reported here show that medium and long chain enoyl-CoA molecules are preferred substrates for MtTFE. Modelling studies indicate how the linear medium and long acyl chains of these substrates can bind to each of the active sites. In addition, crystallographic binding studies have identified three new CoA binding sites which are different from the previously known CoA binding sites of the three TFE active sites. Structure comparisons provide new insights into the properties of ECH, HAD and KAT active sites of MtTFE. The interactions of the adenine moiety of CoA with loop-2 of the ECH active site cause a conformational change of this loop by which a competent ECH active site is formed. The NAD+ binding domain (domain C) of the HAD part of MtTFE has only a few interactions with the rest of the complex and adopts a range of open conformations, whereas the A-domain of the ECH part is rigidly fixed with respect to the HAD part. Two loops, the CB1-CA1 region and the catalytic CB4-CB5 loop, near the thiolase active site and the thiolase dimer interface, have high B-factors. Structure comparisons suggest that a competent and stable thiolase dimer is formed only when complexed with the α-chains, highlighting the importance of the assembly for the proper functioning of the complex