Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
Chemistry. 2020 Sep 4;26(50):11614-11624. doi: 10.1002/chem.202002277. Epub 2020 Aug 7.
Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3-sialyltransferase from Photobacterium phosphoreum JT-ISH-467 (2,3SiaT ) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure-guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaT (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3-Neu5Ac-Lac) from lactose in a one-pot, two-enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new-to-nature sialoconjugates for biological studies.
已定义的唾液酸糖缀合物是研究唾液酸化聚糖在生物系统中作用的重要分子探针。我们表明，来自 Photobacterium phosphoreum JT-ISH-467 的 α2,3-唾液酸转移酶（2,3SiaT）能够容忍唾液酸部分非常广泛的修饰底物范围，包括大酰胺变化、C5/C9 取代和 C5 立体反转。为了降低酶的水解活性，从而降低产物产率，我们进行了广泛的基于结构的突变体研究，鉴定出三种变体，它们对唾液酰基转移的催化效率提高了五倍，对底物水解的效率降低了十倍，对产物水解的效率降低了十倍。变体 2,3SiaT（A151D）在乳糖一锅两步酶级联反应中合成 GM3 三糖（α2,3-Neu5Ac-Lac）时表现出最佳性能。我们的研究表明，可以找到几种互补的解决方案来抑制微生物 GT80 唾液酸转移酶中常见的不期望的水解活性问题。这些新酶是用于合成各种复杂天然和新型唾液酸缀合物的有力催化剂，可用于生物学研究。
