1ss9
From Proteopedia
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| - | [[Image:1ss9.gif|left|200px]] | + | [[Image:1ss9.gif|left|200px]] |
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| - | '''Crystal Structural Analysis of Active Site Mutant Q189E of LgtC''' | + | {{Structure |
| + | |PDB= 1ss9 |SIZE=350|CAPTION= <scene name='initialview01'>1ss9</scene>, resolution 2.60Å | ||
| + | |SITE= | ||
| + | |LIGAND= <scene name='pdbligand=MN:MANGANESE+(II)+ION'>MN</scene> and <scene name='pdbligand=UPF:URIDINE-5'-DIPHOSPHATE-2-DEOXY-2-FLUOROGALACTOSE'>UPF</scene> | ||
| + | |ACTIVITY= [http://en.wikipedia.org/wiki/Lipopolysaccharide_3-alpha-galactosyltransferase Lipopolysaccharide 3-alpha-galactosyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.4.1.44 2.4.1.44] | ||
| + | |GENE= lgtc ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=487 Neisseria meningitidis]) | ||
| + | }} | ||
| + | |||
| + | '''Crystal Structural Analysis of Active Site Mutant Q189E of LgtC''' | ||
| + | |||
==Overview== | ==Overview== | ||
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==About this Structure== | ==About this Structure== | ||
| - | 1SS9 is a [ | + | 1SS9 is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Neisseria_meningitidis Neisseria meningitidis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1SS9 OCA]. |
==Reference== | ==Reference== | ||
| - | Intermediate trapping on a mutant retaining alpha-galactosyltransferase identifies an unexpected aspartate residue., Lairson LL, Chiu CP, Ly HD, He S, Wakarchuk WW, Strynadka NC, Withers SG, J Biol Chem. 2004 Jul 2;279(27):28339-44. Epub 2004 Apr 9. PMID:[http:// | + | Intermediate trapping on a mutant retaining alpha-galactosyltransferase identifies an unexpected aspartate residue., Lairson LL, Chiu CP, Ly HD, He S, Wakarchuk WW, Strynadka NC, Withers SG, J Biol Chem. 2004 Jul 2;279(27):28339-44. Epub 2004 Apr 9. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/15075344 15075344] |
[[Category: Lipopolysaccharide 3-alpha-galactosyltransferase]] | [[Category: Lipopolysaccharide 3-alpha-galactosyltransferase]] | ||
[[Category: Neisseria meningitidis]] | [[Category: Neisseria meningitidis]] | ||
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[[Category: alpha-beta protein]] | [[Category: alpha-beta protein]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 14:08:59 2008'' |
Revision as of 12:09, 20 March 2008
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| , resolution 2.60Å | |||||||
|---|---|---|---|---|---|---|---|
| Ligands: | and | ||||||
| Gene: | lgtc (Neisseria meningitidis) | ||||||
| Activity: | Lipopolysaccharide 3-alpha-galactosyltransferase, with EC number 2.4.1.44 | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
Crystal Structural Analysis of Active Site Mutant Q189E of LgtC
Overview
Lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 alpha-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K(m) for UDP-galactose is not significantly altered, the k(cat) was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 A away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k(cat) value 3000-fold lower than that of the wild type enzyme. A 2.6-A crystal structure of the Q189E mutant with bound uridine 5'-diphospho-2-deoxy-2-fluoro-alpha-d-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate.
About this Structure
1SS9 is a Single protein structure of sequence from Neisseria meningitidis. Full crystallographic information is available from OCA.
Reference
Intermediate trapping on a mutant retaining alpha-galactosyltransferase identifies an unexpected aspartate residue., Lairson LL, Chiu CP, Ly HD, He S, Wakarchuk WW, Strynadka NC, Withers SG, J Biol Chem. 2004 Jul 2;279(27):28339-44. Epub 2004 Apr 9. PMID:15075344
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