2VXM

Screening a Limited Structure-based Library Identifies UDP-GalNAc- Specific Mutants of alpha-1,3 Galactosyltransferase

2VXM の概要

• エントリーDOI: 10.2210/pdb2vxm/pdb
• 関連するPDBエントリー: 1FG5 1G8O 1G93 1GWV 1GWW 1GX0 1GX4 1K4V 1O7O 1O7Q 1VZT 1VZU 1VZX 2JCJ 2JCK 2JCL 2JCO 2VFZ 2VS3 2VS4 2VS5 2VXL
• 分子名称: N-ACETYLLACTOSAMINIDE ALPHA-1,3-GALACTOSYLTRANSFERASE, (4S)-2-METHYL-2,4-PENTANEDIOL, GLYCEROL, ... (4 entities in total)
• 機能のキーワード: glycosyltransferase, 3 galactosyltransferase, transmembrane, golgi apparatus, enzyme mechanism, glycoprotein, metal-binding, signal-anchor, alpha-1, membrane, manganese, transferase, glycosyltransferase galactosyltransferase, transferase substrate specificity
• 由来する生物種: BOS TAURUS (BOVINE)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 128990.95

構造登録者

Tumbale, P.,Jamaluddin, H.,Thiyagarajan, N.,Acharya, K.R.,Brew, K. (登録日: 2008-07-07, 公開日: 2008-09-23, 最終更新日: 2023-12-13)

主引用文献

Tumbale, P.,Jamaluddin, H.,Thiyagarajan, N.,Acharya, K.R.,Brew, K.
Screening a Limited Structure-Based Library Identifies Udp-Galnac-Specific Mutants of {Alpha}-1,3-Galactosyltransferase.
Glycobiology, 18:1036-, 2008

PubMed Abstract: Complex glycans have important roles in biological recognition processes and considerable pharmaceutical potential. The synthesis of novel glycans can be facilitated by engineering glycosyltransferases to modify their substrate specificities. The choice of sites to modify requires the knowledge of the structures of enzyme-substrate complexes while the complexity of protein structures necessitates the exploration of a large array of multisite mutations. The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT), which catalyzes the synthesis of the alpha-Gal epitope, has strict specificity for UDP-galactose as a donor substrate. Based on the structure of a complex of UDP-galactose with alpha3GT, the specificity for the galactose moiety can be partly attributed to residues that interact with the galactose 2-OH group, particularly His280 and Ala282. With the goal of engineering a variant of bovine alpha3GT with GalNAc transferase activity, we constructed a limited library of 456 alpha3GT mutants containing 19 alternative amino acids at position 280, two each at 281 and 282 and six at position 283. Clones (1500) were screened by assaying partially purified bacterially expressed variants for GalNAc transferase activity. Mutants with the highest levels of GalNAc transferase activity, AGGL or GGGL, had substitutions at all four sites. The AGGL mutant had slightly superior GalNAc transferase activity amounting to about 3% of the activity of the wild-type enzyme with UDP-Gal. This mutant had a low activity with UDP-Gal; its crystallographic structure suggests that the smaller side chains at residues 280-282 form a pocket to accommodate the larger acetamido group of GalNAc. Mutational studies indicate that Leu283 is important for stability in this mutant.

PubMed: 18782853
DOI: 10.1093/GLYCOB/CWN083

実験手法

X-RAY DIFFRACTION (2.82 Å)