Structural characterization of a Δ3, Δ2-enoyl-CoA isomerase from Pseudomonas aeruginosa: implications for its involvement in unsaturated fatty acid metabolism.

Gene PA4980 from Pseudomonas aeruginosa encodes a putative enoyl-coenzyme A hydratase/isomerase that is associated with the function of the biofilm dispersion-inducing signal molecule cis-2-decenoic acid. To elucidate the role of PA4980 in cis-2-decenoic acid biosynthesis, we reported the crystal structure of its protein product at 2.39 Å. The structural analysis and substrate binding prediction suggest that it acts as a monofunctional enoyl-coenzyme A isomerase, implicating an alternative pathway of the cis-2-decenoic acid synthesis.

Unsaturated Lipid Assimilation by Mycobacteria Requires Auxiliary cis-trans Enoyl CoA Isomerase.

Mycobacterium tuberculosis (Mtb) can survive in hypoxic necrotic tissue by assimilating energy from host-derived fatty acids. While the expanded repertoire of ß-oxidation auxiliary enzymes is considered crucial for Mtb adaptability, delineating their functional relevance has been challenging. Here, we show that the Mtb fatty acid degradation (FadAB) complex cannot selectively break down cis fatty acyl substrates. We demonstrate that the stereoselective binding of fatty acyl substrates in the Mtb FadB pocket is due to the steric hindrance from Phe287 residue. By developing a functional screen, we classify the family of Mtb Ech proteins as monofunctional or bifunctional enzymes, three of which complement the FadAB complex to degrade cis fatty acids. Crystal structure determination of two cis-trans enoyl coenzyme A (CoA) isomerases reveals distinct placement of active-site residue in Ech enzymes. Our studies thus reveal versatility of Mtb lipid-remodeling enzymes and identify an essential role of stand-alone cis-trans enoyl CoA isomerases in mycobacterial biology.

Structures of yeast peroxisomal Δ(3),Δ(2)-enoyl-CoA isomerase complexed with acyl-CoA substrate analogues: the importance of hydrogen-bond networks for the reactivity of the catalytic base and the oxyanion hole.

Δ(3),Δ(2)-Enoyl-CoA isomerases (ECIs) catalyze the shift of a double bond from 3Z- or 3E-enoyl-CoA to 2E-enoyl-CoA. ECIs are members of the crotonase superfamily. The crotonase framework is used by many enzymes to catalyze a wide range of reactions on acyl-CoA thioesters. The thioester O atom is bound in a conserved oxyanion hole. Here, the mode of binding of acyl-CoA substrate analogues to peroxisomal Saccharomyces cerevisiae ECI (ScECI2) is described. The best defined part of the bound acyl-CoA molecules is the 3',5'-diphosphate-adenosine moiety, which interacts with residues of loop 1 and loop 2, whereas the pantetheine part is the least well defined. The catalytic base, Glu158, is hydrogen-bonded to the Asn101 side chain and is further hydrogen-bonded to the side chain of Arg100 in the apo structure. Arg100 is completely buried in the apo structure and a conformational change of the Arg100 side chain appears to be important for substrate binding and catalysis. The oxyanion hole is formed by the NH groups of Ala70 (loop 2) and Leu126 (helix 3). The O atoms of the corresponding peptide units, Gly69 O and Gly125 O, are both part of extensive hydrogen-bond networks. These hydrogen-bond networks are a conserved feature of the crotonase oxyanion hole and their importance for catalysis is discussed.

Human Δ³,Δ²-enoyl-CoA isomerase, type 2: a structural enzymology study on the catalytic role of its ACBP domain and helix-10.

The catalytic domain of the trimeric human Δ(3),Δ(2)-enoyl-CoA isomerase, type 2 (HsECI2), has the typical crotonase fold. In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the 3E- or 3Z-enoyl-CoA substrate molecules. A catalytic glutamate is essential for the proton transfer between the substrate C2 and C4 atoms for forming the product 2E-enoyl-CoA, which is a key intermediate in the ß-oxidation pathway. The active site is covered by the C-terminal helix-10. In HsECI2, the isomerase domain is extended at its N terminus by an acyl-CoA binding protein (ACBP) domain. Small angle X-ray scattering analysis of HsECI2 shows that the ACBP domain protrudes out of the central isomerase trimer. X-ray crystallography of the isomerase domain trimer identifies the active site geometry. A tunnel, shaped by loop-2 and extending from the catalytic site to bulk solvent, suggests a likely mode of binding of the fatty acyl chains. Calorimetry data show that the separately expressed ACBP and isomerase domains bind tightly to fatty acyl-CoA molecules. The truncated isomerase variant (without ACBP domain) has significant enoyl-CoA isomerase activity; however, the full-length isomerase is more efficient. Structural enzymological studies of helix-10 variants show the importance of this helix for efficient catalysis. Its hydrophobic side chains, together with residues from loop-2 and loop-4, complete a hydrophobic cluster that covers the active site, thereby fixing the thioester moiety in a mode of binding competent for efficient catalysis.

A chiral high-performance liquid chromatography-tandem mass spectrometry method for the stereospecific analysis of enoyl-coenzyme A hydratases/isomerases.

The enzymes catalyzing the stereospecific hydration of 2-enoyl-CoA into the corresponding S- or R-3-hydroxyacyl-CoA are named enoyl-CoA hydratases (ECH), where the S-specific is called ECH-1 and the R-specific is called ECH-2. Current ECH assays are mostly based on spectrophotometric methods. Amongst many limitations, these methods do not directly measure the 3-hydroxyacyl-CoA produced, neither do they allow determination of its stereospecific configuration. We have developed a chiral HPLC method coupled with tandem mass spectrometry (MS) for the sensitive, direct, stereospecific and quantitative analysis of ECH-1/-2 reaction products, or R-/S-3-hydroxyalkanoates in general. The method is based on the reaction of the 3-hydroxyl group on the chiral carbon with 3,5-dimethylphenyl isocyanate, creating a urethane derivative which is then chirally resolved on a chiral HPLC column having 3,5-dimethylphenyl carbamate-derivatized cellulose as the chiral stationary phase. The resolved urethane derivatives are detected using tandem MS in the multiple reactions monitoring (MRM) negative electrospray ionization mode by monitoring the free hydroxy fatty acid fragment ion liberated from its parent urethane derivative. The method resolves the R-/S-enantiomers of 3-hydroxy fatty acid homologues ranging from C6 to C16. Using this method, the net ECH activity present in clarified cell lysates of the bacterium Pseudomonas aeruginosa cultivated in a rich medium was found to be of both ECH-1 and ECH-2. Interestingly, the clarified cell lysate of Escherichia coli cultivated also in a rich medium displayed mainly an ECH-1 (S-specific) activity. This method will facilitate the quantification and stereospecific characterization of ECHs, as well as the chiral lipid profiling of bacterial secondary metabolites containing hydroxyalkanoic acid moieties.