The α/β hydrolase fold

We have identified a new protein fold-the α/β hydrolase fold-that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an α/β sheet, not barrel, of eight β-sheets connected by α-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the α/β hydrolase fold enzymes

Crystal structure of haloalkane dehalogenase: an enzyme to detoxify halogenated alkanes

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 converts 1-haloalkanes to the corresponding alcohols and halide ions with water as the sole cosubstrate and without any need for oxygen or cofactors. The three-dimensional structure has been determined by multiple isomorphous replacement techniques using three heavy atom derivatives. The structure has been refined at 2.4 Å resolution to an R-factor of 17.9%. The monomeric enzyme is a spherical molecule and is composed to two domains: domain I has an α/β type structure with a central eight-stranded mainly parallel β-sheet. Domain II lies like a cap on top of domain I and consists of α-helices connected by loops. Except for the cap domain the structure resembles that of the dienelactone hydrolase in spite of any significant sequence homology. The putative active site is completely buried in an internal hydrophobic cavity which is located between the two domains. From the analysis of the structure it is suggested that Asp124 is the nucleophilic residue essential for the catalysis. It interacts with His289 which is hydrogen-bonded to Asp260.

Refined X-ray Structures of Haloalkane Dehalogenase at pH 6.2 and pH 8.2 and Implications for the Reaction Mechanism

The crystal structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 has been refined at 1.9 Å resolution at two different pH values, the pH of crystallization (pH 6.2) and the pH of optimal activity (pH 8.2), to final R-factors of 16.8% and 16.4%, respectively. Both models show good stereochemical quality. Two non-glycine residues have main-chain torsion angles that are located outside the “allowed” regions in a Ramachandran plot. One of them is the nucleophilic residue Asp124, which, together with the two other active site residues His289 and Asp260, is situated in an internal, predominantly hydrophobic cavity. The other residue, Asn148, helps stabilize the conformations of two of these active-site residues, Asp124 and Asp260. Comparison of the models at pH 6.2 and pH 8.2 revealed one major structural difference. At pH 6.2, a salt-bridge is present between the Nε2 atom of His289 and the Oδ1 atom of Asp124, while at pH 8.2, this salt-bridge is absent, indicating that the Nε2 atom of the histidine residue is mostly deprotonated at the pH of optimum activity. This is in agreement with the putative reaction mechanism in which the Oδ1 atom of Asp124 performs a nucleophilic attack on the substrate, resulting in an intermediate ester. This ester is subsequently cleaved by a hydrolytic water molecule. The high-resolution data sets clearly show the exact position of this water molecule. It is in an ideal position for donating a proton to the Nε2 atom of His289 and subsequently cleaving the covalently bound intermediate ester, releasing the alcohol product. Detailed investigation of both refined models showed a number of unusual structural features. Four out of 11 helices contain an internal proline residue other than in the first turn. Two other α-helices have adopted in their central part a 310 conformation. A novel four-residue turn between a helix and a strand, the αβ4 turn, is located at the site of the bend in the central eight-stranded β-sheet of the dehalogenase structure.

The α/β hydrolase fold

We have identified a new protein fold-the α/β hydrolase fold-that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an α/β sheet, not barrel, of eight β-sheets connected by α-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the α/β hydrolase fold enzymes.