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J. Biol. Chem., Vol. 279, Issue 19, 20501-20510, May 7, 2004

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Reaction Mechanism of Hydroxynitrile Lyases of the /-Hydrolase Superfamily

THE THREE-DIMENSIONAL STRUCTURE OF THE TRANSIENT ENZYME-SUBSTRATE COMPLEX CERTIFIES THE CRUCIAL ROLE OF LYS^236*

Karl Gruber¶, Günter Gartler, Barbara Krammer||, Helmut Schwab||, and Christoph Kratky**

From the Institut für Chemie, Physikalische Chemie, Karl-Franzens Universitaät Heinrichstrasse 28, A-8010 Graz, Austria, the ^||Institut für Molekulare Biotechnologie, Technische Universitaät Graz, Petersgasse 12, A-8010 Graz, Austria, and the Angewandte Biokatalyse Kompetenzzentrum GmbH, Petersgasse 14, A-8010 Graz, Austria

The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the /-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys²³⁶ the catalytic base His²³⁵ would be protonated at neutral pH. All of this suggests that Lys²³⁶ is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN^-, and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of /-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.

Received for publication, February 12, 2004 , and in revised form, February 27, 2004.

The atomic coordinates and structure factors (codes 1sc9, 1sci, 1sck, and 1scq) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Austrian Science Foundation (FWF) through the Spezialforschungsbereich Biokatalyse. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of an APART Fellowship from the Austrian Academy of Sciences.

^** To whom correspondence should be addressed. Tel.: 43-316-380-5417; Fax: 43-316-380-9850; E-mail: Christoph.Kratky{at}uni-graz.at.

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