Crystal Structure of the (R)-Specific Enoyl-CoA Hydratase from Aeromonas caviae Involved in Polyhydroxyalkanoate Biosynthesis

Abstract

The (R)-specific enoyl coenzyme A hydratase ((R)-hydratase) from Aeromonas caviae catalyzes the addition of a water molecule to trans-2-enoyl coenzyme A (CoA), with a chain-length of 4-6 carbons, to produce the corresponding (R)-3-hydroxyacyl-CoA. It forms a dimer of identical subunits with a molecular weight of about 14,000 and is involved in polyhydroxyalkanoate (PHA) biosynthesis. The crystal structure of the enzyme has been determined at 1.5-A resolution. The structure of the monomer consists of a five-stranded antiparallel beta-sheet and a central alpha-helix, folded into a so-called "hot dog" fold, with an overhanging segment. This overhang contains the conserved residues including the hydratase 2 motif residues. In dimeric form, two beta-sheets are associated to form an extended 10-stranded beta-sheet, and the overhangs obscure the putative active sites at the subunit interface. The active site is located deep within the substrate-binding tunnel, where Asp(31) and His(36) form a catalytic dyad. These residues are catalytically important as confirmed by site-directed mutagenesis and are possibly responsible for the activation of a water molecule and the protonation of a substrate molecule, respectively. Residues such as Leu(65) and Val(130) are situated at the bottom of the substrate-binding tunnel, defining the preference of the enzyme for the chain length of the substrate. These results provide target residues for protein engineering, which will enhance the significance of this enzyme in the production of novel PHA polymers. In addition, this study provides the first structural information of the (R)-hydratase family and may facilitate further functional studies for members of the family.

Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation

“…1 c and d). Other reported oligomeric structures of microbial thioesterases include dimers (20,21) and different tetrameric arrangements (14,22,23).…”

Revisiting the Assignment of Rv0241c to Fatty Acid Synthase Type II ofMycobacterium tuberculosis

“…It is a putative (R)-specific enoyl hydratase/3-hydroxyacyl dehydratase belonging to the hydratase 2 protein family and it holds a well-conserved hydratase 2 motif, including the catalytic residues "D-x(4)-H" (5,29). It is predicted to have a "double hot dog fold" structure (5,29), where each of the two structural domains corresponds to a hot dog fold (mainly a long ␣-helix wrapped into a fivestranded antiparallel ␤-sheet [13]). …”

Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

“…Recently, the crystal structure of Aeromonas caviae (R)-specific enoyl-CoA hydratase was resolved, and the catalytic residues were suggested to be Asp-31 and His-36 (11). Also, these amino acids were found to be highly conserved in various (R)-hydratases (11). Multiple alignment of MaoC and PhaJ1 clearly showed that these essential amino acid residues also exist in MaoC at conserved region (Fig.…”

“…These results might have resulted from the presence of various FadB homologous enzymes mentioned above. Recently, the crystal structure of Aeromonas caviae (R)-specific enoyl-CoA hydratase was resolved, and the catalytic residues were suggested to be Asp-31 and His-36 (11). Also, these amino acids were found to be highly conserved in various (R)-hydratases (11).…”