A CRYSTAL STRUCTURE AT A RESOLUTION OF 1.1 A OF THE ENZYME HYDROXYNITRILE LYASE FROM HEVEA BRASILIENSIS SHOULD REVEAL THE FUNCTIONS OF THE CATALYTIC TRIAD RESIDUES

M. Gugganig¹, K.M. Hainbucher¹, J. Zuegg¹, R. J. Morris², V. Lamzin², U.G. Wagner¹, C. Kratky¹

¹Institute of Physical Chemistry, University Graz, Heinrichstr. 28, A-8010 Graz, Austria
²EMBL Hamburg Outstation, DESY, Notkestr. 85, 22603 Hamburg, Germany

It is now a few years since the Hydroxynitrile lyase from Hevea brasiliensis started to attract interest because of its potential applications in industrial biocatalysis. Although in recent years many protein structures of the native Hnl and complexed structures of the Hnl with substrates or inhibitors have been analyzed and many new features of structure-function relationship have emerged, there are still many unanswered questions regarding the protonation state of the catalytic residues and their individual functions.

The Hydroxynitrile lyase from Hevea brasiliensis a member of the a/b hydrolase superfamily reveals a catalytic triad similar to serine hydrolases, consisting of the residues Ser80, His235 and Asp207. In most of the members of the a/b hydrolase family the functions of these catalytic residues are clearly defined. Ser acts as the reaction nucleophile attacking the incoming substrate, whereby histidine acts as the catalytic base in this reaction abstracting a proton from the serine and increasing the nucleophilicity of the residue. The acidic residue (Asp in this case) should stabilize the lowest conformation of the imidazole ring as well as the positive charge on the histidine that is formed during reaction.

Investigations of enzyme inhibitor complexes of the Hydroxynitrile lyase revealed a modified catalytic triad which is separated by a water molecule located between the catalytic residues Ser and His. The active site residue Ser80 is also proposed to react in two different ways in catalysis, namely as a nucleophile and an electrophile. The reason for this bifunctionality of the serine is proposed to be due to a water molecule.

To reveal the definitive protonation state and the functions of the catalytic residues of this potential biocatalyst a crystal structure at a resolution of 1.1 A has been determined.