Glycosylation is one of the most prevalent posttranslational modifications that profoundly affects the structure and functions of proteins in a wide variety of biological recognition events. However, the structural complexity and heterogeneity of glycoproteins, usually resulting from the variations of glycan components and/or the sites of glycosylation, often complicates detailed structure-function relationship studies and hampers the therapeutic applications of glycoproteins. To address these challenges, various chemical and biological strategies have been developed for producing glycan-defined homogeneous glycoproteins. This review highlights recent advances in the development of chemoenzymatic methods for synthesizing homogeneous glycoproteins, including the generation of various glycosynthases for synthetic purposes, endoglycosidase-catalyzed glycoprotein synthesis and glycan remodeling, and direct enzymatic glycosylation of polypeptides and proteins. The scope, limitation, and future directions of each method are discussed.

Immunoglobulin G (IgG) glycosylation critically modulates antibody effector functions. Streptococcus pyogenes secretes a unique endo-β- N -acetylglucosaminidase, EndoS2, which deglycosylates the conserved N -linked glycan at Asn297 on IgG Fc to eliminate its effector functions and evade the immune system. EndoS2 and specific point mutants have been used to chemoenzymatically synthesize antibodies with customizable glycosylation for gain of functions. EndoS2 is useful in these schemes because it accommodates a broad range of N -glycans, including high-mannose, complex, and hybrid types; however, its mechanism of substrate recognition is poorly understood. We present crystal structures of EndoS2 alone and bound to complex and high-mannose glycans; the broad N -glycan specificity is governed by critical loops that shape the binding site of EndoS2. Furthermore, hydrolytic experiments, domain-swap chimeras, and hydrogen–deuterium exchange mass spectrometry reveal the importance of the carbohydrate-binding module in the mechanism of IgG recognition by EndoS2, providing insights into engineering enzymes to catalyze customizable glycosylation reactions.

It was first observed that PPL, lipase from porcine pancreas, and several other lipases have a promiscuous ability to catalyse asymmetric aldol reactions between acetones and aldehydes in the presence of water.Biocatalytic promiscuity, a new frontier extending the use of enzymes in organic synthesis, has attracted much attention and expanded rapidly in recent years. 1 It focuses on the enzyme catalytic activities with unnatural substrates and alternative chemical transformations, such as the side ability harbored by decarboxylase to catalyse acyloin condensation. 2 Exploiting enzyme catalytic promiscuity might lead to improvements in existing catalysts and provide novel synthesis pathways that are currently not available. Some elegant works have been done in the last decades. 3 Among the promiscuous enzymes, hydrolases (such as lipase, protease and esterase) undoubtedly play an important role due to their high stability, wide sources and broad range of substrates. 4 Recently, several promiscuous hydrolase-catalysed reactions have been reported. 5 For instance, Wu et al. demonstrated that penicillin G acylase, a hydrolase which is widely used as a biocatalyst in the enzymatic synthesis of b-lactam antibiotics, can catalyse Markovnikov addition of allopurinol to vinyl ester. 6 A further example is reported by the group of Gotor. 7 They found an unprecedented lipase catalysed Michael addition of secondary amines to acrylonitrile. These cases and other relevant reports encouraged us to believe that the catalytic activities for addition reaction rather than the well-known hydrolytic function may also have a natural role in hydrolase evolution.Aldol addition is one of the most useful methods for carboncarbon bond formation in organic synthesis. 8 Berglund and co-workers once used mutant CAL-B (lipase from Candida antarctica) to catalyse aldol addition in 2003. 9 Although the Ser105Ala mutant CAL-B exhibited an increased reaction rate as compared with the wide type in their experiments, both of them showed quite low activities (reaction time more than 50 days). Besides, the enzymatic process is not enantioselective and only simple aliphatic aldehydes, such as propanal and hexanal, had been used. Generally, practical lipase-catalysed aldol reactions hadn't been developed in organic synthesis. To the best of our knowledge, other lipase-catalysed aldol additions, especially asymmetric aldol reactions have never been reported.

Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multi-step chemical synthesis or the inability of the biosynthetic α1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1,6-fucosynthase and fucoligase for direct core-fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1,6-fucosidase mutants could introduce an α1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide, but also to high-mannose and complex type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.

The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-β-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man 9 GlcNAc 2 Asn substrate complex, and two EndoBT-3987-Man 9 GlcNAc and EndoBT-3987-Man 5 GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.