1sci
From Proteopedia
(New page: 200px<br /><applet load="1sci" size="450" color="white" frame="true" align="right" spinBox="true" caption="1sci, resolution 2.18Å" /> '''K236L mutant of hydr...) |
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| - | [[Image:1sci.jpg|left|200px]]<br /><applet load="1sci" size=" | + | [[Image:1sci.jpg|left|200px]]<br /><applet load="1sci" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1sci, resolution 2.18Å" /> | caption="1sci, resolution 2.18Å" /> | ||
'''K236L mutant of hydroxynitrile lyase from Hevea brasiliensis'''<br /> | '''K236L mutant of hydroxynitrile lyase from Hevea brasiliensis'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from | + | 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. |
==About this Structure== | ==About this Structure== | ||
| - | 1SCI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hevea_brasiliensis Hevea brasiliensis] with SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Transfered_to_EC_4.1.2.37 Transfered to EC 4.1.2.37], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.1.2.39 4.1.2.39] Full crystallographic information is available from [http:// | + | 1SCI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hevea_brasiliensis Hevea brasiliensis] with <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Transfered_to_EC_4.1.2.37 Transfered to EC 4.1.2.37], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.1.2.39 4.1.2.39] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1SCI OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Hevea brasiliensis]] | [[Category: Hevea brasiliensis]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Transfered to EC 4 | + | [[Category: Transfered to EC 4 1.2 37]] |
[[Category: Gartler, G.]] | [[Category: Gartler, G.]] | ||
[[Category: Gruber, K.]] | [[Category: Gruber, K.]] | ||
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[[Category: catalytic triad]] | [[Category: catalytic triad]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:00:06 2008'' |
Revision as of 13:00, 21 February 2008
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K236L mutant of hydroxynitrile lyase from Hevea brasiliensis
Overview
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.
About this Structure
1SCI is a Single protein structure of sequence from Hevea brasiliensis with as ligand. Active as Transfered to EC 4.1.2.37, with EC number 4.1.2.39 Full crystallographic information is available from OCA.
Reference
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., Gruber K, Gartler G, Krammer B, Schwab H, Kratky C, J Biol Chem. 2004 May 7;279(19):20501-10. Epub 2004 Mar 3. PMID:14998991
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