1SCI

K236L mutant of hydroxynitrile lyase from Hevea brasiliensis

Summary for 1SCI

• Entry DOI: 10.2210/pdb1sci/pdb
• Related: 1SC9 1SCK 1SCQ 1qj4 1yas 2yas 3yas 4yas 5yas 6yas 7yas
• Descriptor: (S)-acetone-cyanohydrin lyase, SULFATE ION (3 entities in total)
• Functional Keywords: alpha-beta hydrolase fold, catalytic triad, lyase
• Biological source: Hevea brasiliensis
• Total number of polymer chains: 1
• Total formula weight: 29630.83

Authors

Gruber, K.,Gartler, G.,Krammer, B.,Schwab, H.,Kratky, C. (deposition date: 2004-02-12, release date: 2004-06-29, Last modification date: 2023-10-25)

Primary citation

Gruber, K.,Gartler, G.,Krammer, B.,Schwab, H.,Kratky, C.
Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236
J.Biol.Chem., 279:20501-20510, 2004

PubMed Abstract: The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-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 Lys(236) the catalytic base His(235) would be protonated at neutral pH. All of this suggests that Lys(236) 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 alpha/beta-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.

PubMed: 14998991
DOI: 10.1074/jbc.M401575200

Experimental method

X-RAY DIFFRACTION (2.18 Å)