Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, inc luding trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-O gamm a. All other complexes showed the substrate or inhibitor molecule merely hy drogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, elimin ating previous uncertainties concerning residual electron density within th e active site, and leading to the observation of two conserved water molecu les. One of them was found to be conserved in all complex structures and ap pears to have mainly structural significance. The other water molecule is c onserved in all structures except for the complex with rhodanide; it is hyd rogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest imp lications for the enzyme mechanism. It appears that the enzyme-catalyzed cy anohydrin formation is unlikely to proceed via a hemiacetal or hemiketal in termediate covalently attached to the enzyme, despite the observation of su ch an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activa ted by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hyd roxy groups. A hydrogen cyanide molecule subsequently replaces a water mole cule and is deprotonated presumably by the His235 base. Deprotonation is fa cilitated by the proximity of the positive charge of the Lys236 side chain.