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1yro
From Proteopedia
(New page: 200px<br /><applet load="1yro" size="450" color="white" frame="true" align="right" spinBox="true" caption="1yro, resolution 1.90Å" /> '''Crystal structure of...) |
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| - | [[Image:1yro.gif|left|200px]]<br /><applet load="1yro" size=" | + | [[Image:1yro.gif|left|200px]]<br /><applet load="1yro" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1yro, resolution 1.90Å" /> | caption="1yro, resolution 1.90Å" /> | ||
'''Crystal structure of beta14,-galactosyltransferase mutant ARG228Lys in complex with alpha-lactalbumin in the presence of UDP-galactose and Mn'''<br /> | '''Crystal structure of beta14,-galactosyltransferase mutant ARG228Lys in complex with alpha-lactalbumin in the presence of UDP-galactose and Mn'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Beta-1,4-galactosyltransferase I (beta4Gal-T1) normally transfers Gal from | + | Beta-1,4-galactosyltransferase I (beta4Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn(2+) ion (Gal-T activity) and also transfers Glc from UDP-Glc to GlcNAc (Glc-T activity), albeit at only 0.3% efficiency. In addition, alpha-lactalbumin (LA) enhances this Glc-T activity more than 25 times. Comparison of the crystal structures of UDP-Gal- and UDP-Glc-bound beta4Gal-T1 reveals that the O4 hydroxyl group in both Gal and Glc moieties forms a hydrogen bond with the side chain carboxylate group of Glu317. The orientation of the O4 hydroxyl of glucose causes a steric hindrance to the side chain carboxylate group of Glu317, accounting for the enzyme's low Glc-T activity. In this study, we show that mutation of Arg228, a residue in the vicinity of Glu317, to lysine (R228K-Gal-T1) results in a 15-fold higher Glc-T activity, which is further enhanced by LA to nearly 25% of the Gal-T activity of the wild type. The kinetic parameters indicate that the main effect of the mutation of Arg228 to lysine is on the k(cat) of Glc-T, which increases 3-4-fold, both in the absence and in the presence of LA; simultaneously, the k(cat) for the Gal-T reaction is reduced 30-fold. The crystal structure of R228K-Gal-T1 complexed with LA, UDP-Gal, and Mn(2+) determined at 1.9 A resolution shows that the Asp318 side chain exhibits a minor alternate conformation, compared to that in the wild type. This alternate conformation now causes a steric hindrance to the O4 hydroxyl group of the Gal moiety of UDP-Gal, probably causing the dissociation of UDP-Gal and the reduced k(cat) of the Gal-T reaction. |
==About this Structure== | ==About this Structure== | ||
| - | 1YRO is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] and [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus] with GDU, CA, MN, UDP, MES and PG4 as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http:// | + | 1YRO is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] and [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus] with <scene name='pdbligand=GDU:'>GDU</scene>, <scene name='pdbligand=CA:'>CA</scene>, <scene name='pdbligand=MN:'>MN</scene>, <scene name='pdbligand=UDP:'>UDP</scene>, <scene name='pdbligand=MES:'>MES</scene> and <scene name='pdbligand=PG4:'>PG4</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1YRO OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Protein complex]] | [[Category: Protein complex]] | ||
[[Category: Boeggeman, E.]] | [[Category: Boeggeman, E.]] | ||
| - | [[Category: Qasba, P | + | [[Category: Qasba, P K.]] |
[[Category: Ramakrishnan, B.]] | [[Category: Ramakrishnan, B.]] | ||
[[Category: CA]] | [[Category: CA]] | ||
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[[Category: arg228lys mutation; udp-gal complex]] | [[Category: arg228lys mutation; udp-gal complex]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:08:23 2008'' |
Revision as of 14:08, 21 February 2008
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Crystal structure of beta14,-galactosyltransferase mutant ARG228Lys in complex with alpha-lactalbumin in the presence of UDP-galactose and Mn
Overview
Beta-1,4-galactosyltransferase I (beta4Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn(2+) ion (Gal-T activity) and also transfers Glc from UDP-Glc to GlcNAc (Glc-T activity), albeit at only 0.3% efficiency. In addition, alpha-lactalbumin (LA) enhances this Glc-T activity more than 25 times. Comparison of the crystal structures of UDP-Gal- and UDP-Glc-bound beta4Gal-T1 reveals that the O4 hydroxyl group in both Gal and Glc moieties forms a hydrogen bond with the side chain carboxylate group of Glu317. The orientation of the O4 hydroxyl of glucose causes a steric hindrance to the side chain carboxylate group of Glu317, accounting for the enzyme's low Glc-T activity. In this study, we show that mutation of Arg228, a residue in the vicinity of Glu317, to lysine (R228K-Gal-T1) results in a 15-fold higher Glc-T activity, which is further enhanced by LA to nearly 25% of the Gal-T activity of the wild type. The kinetic parameters indicate that the main effect of the mutation of Arg228 to lysine is on the k(cat) of Glc-T, which increases 3-4-fold, both in the absence and in the presence of LA; simultaneously, the k(cat) for the Gal-T reaction is reduced 30-fold. The crystal structure of R228K-Gal-T1 complexed with LA, UDP-Gal, and Mn(2+) determined at 1.9 A resolution shows that the Asp318 side chain exhibits a minor alternate conformation, compared to that in the wild type. This alternate conformation now causes a steric hindrance to the O4 hydroxyl group of the Gal moiety of UDP-Gal, probably causing the dissociation of UDP-Gal and the reduced k(cat) of the Gal-T reaction.
About this Structure
1YRO is a Protein complex structure of sequences from Bos taurus and Mus musculus with , , , , and as ligands. Full crystallographic information is available from OCA.
Reference
Mutation of arginine 228 to lysine enhances the glucosyltransferase activity of bovine beta-1,4-galactosyltransferase I., Ramakrishnan B, Boeggeman E, Qasba PK, Biochemistry. 2005 Mar 8;44(9):3202-10. PMID:15736931
Page seeded by OCA on Thu Feb 21 16:08:23 2008
Categories: Bos taurus | Mus musculus | Protein complex | Boeggeman, E. | Qasba, P K. | Ramakrishnan, B. | CA | GDU | MES | MN | PG4 | UDP | Arg228lys mutation; udp-gal complex
