Abstract
Endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the beta1,4 linkage of N,N'-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the Km value of Y217F for an acceptor substrate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175"knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-beta-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.
Highlights
We describe two interesting mutants of Endo--N-acetylglucosaminidase from Mucor hiemalis (Endo-M); one shows significantly enhanced transglycosylation activity, and the other shows glycosynthase-like activity when the activated sugar oxazoline is used as the donor substrate
Materials—The biantennary complex-type sialylglycopeptide (SGP), Lys-Val-Ala-Asn((NeuAc-Gal-GlcNAc-Man)2ManGlcNAc2)-Lys-Thr was prepared from hen egg yolks following the reported procedure [13, 25]; Man9GlcNAc2-Asn was prepared from soy bean flour according to our modified procedure [26]; the GlcNAc-pentapeptide (Glu-Asn(GlcNAc)-Ile-ThrVal) [1] derived from erythropoietin and a 34-mer glycopeptide (C34) (Trp-Met-Glu-TrpAsp-Arg-Glu-Ile-Asn-Asn(GlcNAc)-Tyr-Thr-Ser-Leu-Ile-GluGlu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu) [9] derived from HIV-1 gp41 were prepared by automatic solidphase peptide synthesis using an N-(9-fluorenyl)methoxycarbonyl approach [18]. 4-Methylumbelliferyl--D-N-acetylglucosaminide (4MU-GlcNAc) and chitinase from S. griseus were purchased from Sigma
In the case of the mutant Y217F, the rate of the total Compound 5 was treated with BF31⁄7Et2O and trimethylsilyl consumption of the donor substrate SGP was much slower than bromide in the presence of collidine to provide the correspondthat of the wild type, and the yield of the transglycosylation ing O-acetylated sugar oxazoline derivative, which was subject product continuously increased over 400 min
Summary
Materials—The biantennary complex-type sialylglycopeptide (SGP), Lys-Val-Ala-Asn((NeuAc-Gal-GlcNAc-Man)2ManGlcNAc2)-Lys-Thr was prepared from hen egg yolks following the reported procedure [13, 25]; Man9GlcNAc2-Asn was prepared from soy bean flour according to our modified procedure [26]; the GlcNAc-pentapeptide (Glu-Asn(GlcNAc)-Ile-ThrVal) [1] derived from erythropoietin (amino acid sequence 37– 41) and a 34-mer glycopeptide (C34) (Trp-Met-Glu-TrpAsp-Arg-Glu-Ile-Asn-Asn(GlcNAc)-Tyr-Thr-Ser-Leu-Ile-GluGlu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu) [9] derived from HIV-1 gp were prepared by automatic solidphase peptide synthesis using an N-(9-fluorenyl)methoxycarbonyl approach [18]. 4-Methylumbelliferyl--D-N-acetylglucosaminide (4MU-GlcNAc) and chitinase from S. griseus were purchased from Sigma. The preliminary structure suggests that the residues Asn171, Tyr205, Trp216, Asn218, Asn242, Phe243, and Trp244 may locate around the catalytic proton donor Glu173 and interact with substrate within a putative catalytic cleft of Endo-A.4 Based on this information, we have carried out site-directed mutagenesis of Synthesis of HIV-1 gp Glycopeptide Man9GlcNAc2-C34 the corresponding residues in Endo-M and assayed the hydrol[10] Using Mutant N175A—To a solution of GlcNAc-C34 [9] ysis and transglycosylation activities of these mutants (Table 1). The mixture was lyophilized, and the residue was subject to preparative HPLC to give the product Man9GlcNAc2-C34 [10] (0.49 mg, 72%): analytical HPLC, tR ϭ 17.2 min (linear gradient of 0 –90% MeCN containing 0.1% trifluoroacetic acid in 25 min, flow rate 1 ml/min); ESI-MS: calculated M ϭ 6155.5; found, 1540.5 [M ϩ 4H]4ϩ, 1499.6 [M-Man ϩ 4H]4ϩ, 1232.3 [M ϩ 5H]5ϩ, 1200.0 [M-Man ϩ 5H]5ϩ.
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