Insight Into Substrate Recognition Sites Of Endo-β-N-acetylglucosaminidase From Ogataea Minuta

Endo-β-N-acetylglucosaminidase(ENGase) is defined as the enzyme hydrolyzing the glycosidic bond in N, N’-diacetylchitobiose of N-glycans. Basically, an ENGase belongs to glycoside hydrolase family 18(GH18) or glycoside hydrolase family 85(GH85). The GH18 ENGase, such as Endo-H, could catalyze the glycan hydrolysis. O n the other hand, GH85 enzymes such as Endo-A and Endo-M, have the attractive feature to transfer various oligosaccharides onto GlcNAc acceptor except for their hydrolytic activity. The transglycosylation activity promises the GH85 ENGase to be a valuable tool for chemo-enzymatic synthesis of glycoproteins. In this work, I focused on an ENGase from methylotrophic yeast Ogataea minuta, named Endo-Om, which is the member of GH85.Endo-Om is a non-glycosylated cytosolic protein of yeast. For the expression of functional non- glycosylated proteins, Escherichia coli(E. coli) is always considered as a better organism, since it possesses the advantages including rapid growth, quick expression and varieties of expression vectors and genetically modified host strains. Herein, we show the expression of Endo-O m by using E. coli. At the beginning, the recombinant Endo-Om was able to be overexpressed by E. coli BL21(DE3)pLySs, but mainly remained in the insoluble fraction. We then optimized the culture condition and obtained the soluble form of functional Endo-Om. Using the purified Endo-O m, the hydrolytic activity against fluorescent- labeled oligosaccharides was examined.Despite of their potential application to the chemo-enzymatic synthesis of glycoconjugates, very little is currently known about the oligosaccharide substrate recognition mechanism of ENGases. For Endo-Om, preliminary structural modeling experiments predicted that Trp-295 forms an important interaction with the α-1,3-linked mannose residue in the trimannosyl core of N-glycan rather than being directly involved in the catalytic activity. Thus, we produced the mutagenesis of Endo-Om at Trp-295 to investigate its effect on hydrolytic activity. Notably, the Trp residue is highly conserved only in eukaryotic ENGases. When Trp mutated to Ala(A), Glu(E), Gln(Q), the mutants lose their activity. Additionally, comparing with wild-type Endo-Om, the W295 F mutation resulted in a 2-3-fold increase of the hydrolytic activity using pyridylaminated oligosaccharides and glycoproteins as substrates. Moreover, the W295 Y mutation exhibited increased hydrolytic activity only towards agalacto-biantennary oligosaccharide. It could be concluded that an aromatic amino acid is required at position 295. This study investigated the substrate recognition mechanism of Endo-Om, which will facilitate the application of this enzyme in the protein engineering.