The 3D structures of complexes between the hydroxynitrile
lyase from Hevea brasiliensis (Hb–HNL) and
several substrate and/or inhibitor molecules, including
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-Oγ.
All other complexes showed the substrate or inhibitor molecule
merely hydrogen bonded to the protein. In addition, the
native crystal structure of Hb–HNL was redetermined
at cryo-temperature and at room temperature, eliminating
previous uncertainties concerning residual electron density
within the active site, and leading to the observation
of two conserved water molecules. One of them was found
to be conserved in all complex structures and appears to
have mainly structural significance. The other water molecule
is conserved in all structures except for the complex with
rhodanide; it is hydrogen bonded to the imidazole of the
catalytic His235 and appears to affect the Hb–HNL
catalyzed reaction. The observed 3D structural data suggest
implications for the enzyme mechanism. It appears that
the enzyme-catalyzed cyanohydrin formation is unlikely
to proceed via a hemiacetal or hemiketal intermediate covalently
attached to the enzyme, despite the observation of such
an intermediate for the complex with trichloracetaldehyde.
Instead, the data are consistent with a mechanism where
the incoming substrate is activated by hydrogen bonding
with its carbonyl oxygen to the Ser80 and Thr11 hydroxy
groups. A hydrogen cyanide molecule subsequently replaces
a water molecule and is deprotonated presumably by the
His235 base. Deprotonation is facilitated by the proximity
of the positive charge of the Lys236 side chain.