Tumbale, Percy and Jamaluddin, Haryati and Thiyagarajan, Nethaji and Acharya, K. Ravi and Brew, Keith (2008) Screening a limited structure-based library identifies UDP-GalNAc-specific mutants of -1,3 galactosyltransferase. Glycobiology . ISSN 0959-6658
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Official URL: http://dx.doi.org/10.1093/glycob/cwn083
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 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, Î±-1,3 galactosyltransferase (Î±3GT), which catalyzes the synthesis of the Î±-gal epitope, has strict specificity for UDP-galactose as a donor substrate. Based on structure of a complex of UDP-galactose with Î±3GT, 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 Î±3GT with GalNAc transferase activity, we constructed a limited library of 456 Î±3GT mutants containing 19 alternative amino acids at position 280, two each at 281 and 282 and 6 at position 283. Clones (1500) were screened by assaying partially purified bacterially expressed variants for GalNAc transferase activity. Mutants with highest levels of GalNAc transferase activity had substitutions at all four sites, AGGL or GGGL. 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.
|Uncontrolled Keywords:||combinatorial library, crystallographic structure, glycosyltransferase mutagenesis, specificity|
|Subjects:||Q Science > QH Natural history > QH301 Biology|
|Deposited By:||Ms Zalinda Shuratman|
|Deposited On:||20 Nov 2008 06:50|
|Last Modified:||20 Nov 2008 06:50|
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