Tumbale Percy, Jamaluddin Haryati, Thiyagarajan Nethaji, Acharya K Ravi, Brew Keith
Department of Biomedical Science, College of Biomedical Science, Florida Atlantic University, Glades Road, Boca Raton, FL 33431, USA.
Glycobiology. 2008 Dec;18(12):1036-43. doi: 10.1093/glycob/cwn083. Epub 2008 Sep 9.
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.
复杂聚糖在生物识别过程中发挥着重要作用，并且具有可观的药用潜力。通过对糖基转移酶进行工程改造以改变其底物特异性，可促进新型聚糖的合成。选择修饰位点需要了解酶-底物复合物的结构，而蛋白质结构的复杂性则需要探索大量的多位点突变。保留型糖基转移酶α-1,3-半乳糖基转移酶（α3GT）催化α-Gal表位的合成，对作为供体底物的UDP-半乳糖具有严格的特异性。基于UDP-半乳糖与α3GT复合物的结构，对半乳糖部分的特异性可部分归因于与半乳糖2-OH基团相互作用的残基，特别是His280和Ala282。为了构建具有GalNAc转移酶活性的牛α3GT变体，我们构建了一个包含456个α3GT突变体的有限文库，在第280位含有19种替代氨基酸，第281和282位各有两种，第283位有六种。通过检测部分纯化的细菌表达变体的GalNAc转移酶活性，筛选出1500个克隆。具有最高GalNAc转移酶活性水平的突变体AGGL或GGGL在所有四个位点都有替换。AGGL突变体的GalNAc转移酶活性略高，约为野生型酶与UDP-Gal活性的3%。该突变体对UDP-Gal的活性较低；其晶体结构表明，第280 - 282位残基处较小的侧链形成一个口袋，以容纳GalNAc较大的乙酰氨基基团。突变研究表明，Leu283对该突变体的稳定性很重要。
