1yro

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Revision as of 04:53, 21 November 2007

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spinqualitylabelsall models 1yro, resolution 1.90Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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 GDU, CA, MN, UDP, MES and PG4 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

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