Glycoside Phosphorylases Research Articles

CAZac: an activity descriptor for carbohydrate-active enzymes.

The Carbohydrate-Active enZYme database (CAZy; www.cazy.org) has been providing the reference classification of carbohydrate-active enzymes (CAZymes) for >30 years. Based on literature survey, the sequence-based families of CAZymes are enriched with functional data by using the International Union of Biochemistry and Molecular Biology Enzyme Commission (EC) number system. However, this system was not developed to search or compare functional information. To better harness functional information, we have developed CAZac (CAZyme activity descriptor), a multicriterion system that describes CAZymes' mechanisms, glycosidic bond orientations, subsites and inter-residue connectivities. This new system, implemented for glycoside hydrolases, glycoside phosphorylases, transglycosidases, polysaccharide lyases and lytic polysaccharide monooxygenases allows complex searches in the CAZy database to uncover the evolution of substrate specificity and mechanisms of CAZymes across families.

Glycoside Phosphorylase Catalyzed Cellulose and β-1,3-Glucan Synthesis Using Chromophoric Glycosyl Acceptors.

Glycoside phosphorylases are enzymes that are frequently used for polysaccharide synthesis. Some of these enzymes have broad substrate specificity, enabling the synthesis of reducing-end-functionalized glucan chains. Here, we explore the potential of glycoside phosphorylases in synthesizing chromophore-conjugated polysaccharides using commercially available chromophoric model compounds as glycosyl acceptors. Specifically, we report cellulose and β-1,3-glucan synthesis using 2-nitrophenyl β-d-glucopyranoside, 4-nitrophenyl β-d-glucopyranoside, and 2-methoxy-4-(2-nitrovinyl)phenyl β-d-glucopyranoside with Clostridium thermocellum cellodextrin phosphorylase and Thermosipho africanus β-1,3-glucan phosphorylase as catalysts. We demonstrate activity for both enzymes with all assayed chromophoric acceptors and report the crystallization-driven precipitation and detailed structural characterization of the synthesized polysaccharides, i.e., their molar mass distributions and various structural parameters, such as morphology, fibril diameter, lamellar thickness, and crystal form. Our results provide insights for the studies of chromophore-conjugated low molecular weight polysaccharides, glycoside phosphorylases, and the hierarchical assembly of crystalline cellulose and β-1,3-glucan.

Converting Bulk Sugars into Functional Fibers: Discovery and Application of a Thermostable β-1,3-Oligoglucan Phosphorylase.

Despite their broad application potential, the widespread use of β-1,3-glucans has been hampered by the high cost and heterogeneity associated with current production methods. To address this challenge, scalable and economically viable processes are needed for the production of β-1,3-glucans with tailorable molecular mass distributions. Glycoside phosphorylases have shown to be promising catalysts for the bottom-up synthesis of β-1,3-(oligo)glucans since they combine strict regioselectivity with a cheap donor substrate (i.e., α-glucose 1-phosphate). However, the need for an expensive priming substrate (e.g., laminaribiose) and the tendency to produce shorter oligosaccharides still form major bottlenecks. Here, we report the discovery and application of a thermostable β-1,3-oligoglucan phosphorylase originating from Anaerolinea thermophila (AtβOGP). This enzyme combines a superior catalytic efficiency toward glucose as a priming substrate, high thermostability, and the ability to synthesize high molecular mass β-1,3-glucans up to DP 75. Coupling of AtβOGP with a thermostable variant of Bifidobacterium adolescentis sucrose phosphorylase enabled the efficient production of tailorable β-1,3-(oligo)glucans from sucrose, with a near-complete conversion of >99 mol %. This cost-efficient process for the conversion of renewable bulk sugar into β-1,3-(oligo)glucans should facilitate the widespread application of these versatile functional fibers across various industries.

Substrate-Assisted Mechanism for the Degradation of N-Glycans by a Gut Bacterial Mannoside Phosphorylase

The unknown human gut bacterium mannoside phosphorylase (UhgbMP) is involved in the metabolization of eukaryotic N-glycans lining the intestinal epithelium, a factor associated with the onset and symptoms of inflammatory bowel diseases. In contrast with most glycoside phosphorylases, the putative catalytic acid of UhgbMP, Asp104, is far from the scissile glycosidic bond, challenging the classical Koshland mechanism. Using quantum mechanics/molecular mechanics metadynamics, we demonstrate that the enzyme operates by substrate-assisted catalysis via the 3-hydroxyl group of the mannosyl unit, following a 1S5/B2,5 → [B2,5]‡ → 0S2 conformational itinerary. Given the conservation of the active site hydrogen bond network across the family, this mechanism is expected to apply to other GH130 enzymes, as well as recently characterized mannoside phosphorylases with similar folds. Gaining insight into the catalytic reaction of these enzymes can aid the design of specific inhibitors to control interactions between gut microbes and the host.

A Synthetic Gene Library Yields a Previously Unknown Glycoside Phosphorylase That Degrades and Assembles Poly-β-1,3-GlcNAc, Completing the Suite of β-Linked GlcNAc Polysaccharides.

The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally characterized members continues to grow. This wealth of sequence data presented an exciting opportunity to explore the ever-expanding CAZy DB to discover new GPs with never-before-described functionalities. Utilizing an in silico sequence analysis of CAZy family GH94, we discovered and then functionally and structurally characterized the new GP β-1,3-N-acetylglucosaminide phosphorylase. This new GP was sourced from the genome of the cell-wall-less Mollicute bacterium, Acholeplasma laidlawii and was found to synthesize β-1,3-linked N-acetylglucosaminide linkages. The resulting poly-β-1,3-N-acetylglucosamine represents a new, previously undescribed biopolymer that completes the set of possible β-linked GlcNAc homopolysaccharides together with chitin (β-1,4) and PNAG (poly-β-1,6-N-acetylglucosamine). The new biopolymer was denoted acholetin, a combination of the genus Acholeplasma and the polysaccharide chitin, and the new GP was thus denoted acholetin phosphorylase (AchP). Use of the reverse phosphorolysis action of AchP provides an efficient method to enzymatically synthesize acholetin, which is a new biodegradable polymeric material.

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases.

Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction.

Discovery of solabiose phosphorylase and its application for enzymatic synthesis of solabiose from sucrose and lactose

Glycoside phosphorylases (GPs), which catalyze the reversible phosphorolysis of glycosides, are promising enzymes for the efficient production of glycosides. Various GPs with new catalytic activities are discovered from uncharacterized proteins phylogenetically distant from known enzymes in the past decade. In this study, we characterized Paenibacillus borealis PBOR_28850 protein, belonging to glycoside hydrolase family 94. Screening of acceptor substrates for reverse phosphorolysis, in which α-d-glucose 1-phosphate was used as the donor substrate, revealed that the recombinant PBOR_28850 produced in Escherichia coli specifically utilized d-galactose as an acceptor and produced solabiose (β-d-Glcp-(1 → 3)-d-Gal). This indicates that PBOR_28850 is a new GP, solabiose phosphorylase. PBOR_28850 catalyzed the phosphorolysis and synthesis of solabiose through a sequential bi-bi mechanism involving the formation of a ternary complex. The production of solabiose from lactose and sucrose has been established. Lactose was hydrolyzed to d-galactose and d-glucose by β-galactosidase. Phosphorolysis of sucrose and synthesis of solabiose were then coupled by adding sucrose, sucrose phosphorylase, and PBOR_28850 to the reaction mixture. Using 210 mmol lactose and 280 mmol sucrose, 207 mmol of solabiose was produced. Yeast treatment degraded the remaining monosaccharides and sucrose without reducing solabiose. Solabiose with a purity of 93.7% was obtained without any chromatographic procedures.

Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases

Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu472 was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu616 instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family.

Correction to “Facile Enzymatic Synthesis of Diverse Naturally Occurring β-d-Mannopyranosides Catalyzed by Glycoside Phosphorylases”

ADVERTISEMENT RETURN TO ISSUEPREVCorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to “Facile Enzymatic Synthesis of Diverse Naturally Occurring β-d-Mannopyranosides Catalyzed by Glycoside Phosphorylases”Jing LiuJing LiuMore by Jing Liu, Xuefei YinXuefei YinMore by Xuefei Yin, Zitao LiZitao LiMore by Zitao Li, Xiaocong WuXiaocong WuMore by Xiaocong Wu, Zhaoxuan ZhengZhaoxuan ZhengMore by Zhaoxuan Zheng, Junqiang FangJunqiang FangMore by Junqiang Fang, Guofeng GuGuofeng GuMore by Guofeng Guhttps://orcid.org/0000-0002-0969-7487, Peng G. WangPeng G. WangMore by Peng G. Wanghttps://orcid.org/0000-0003-3335-6794, and Xianwei Liu*Xianwei Liu*E-mail: [email protected]More by Xianwei Liuhttps://orcid.org/0000-0002-0182-535XCite this: ACS Catal. 2021, 11, 21, 13706Publication Date (Web):October 27, 2021Publication History Published online27 October 2021Published inissue 5 November 2021https://doi.org/10.1021/acscatal.1c04756Copyright © 2021 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views449Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (597 KB) Get e-AlertsSUBJECTS:Addition reactions,Carbohydrates,Filtration,Gels,Mixtures Get e-Alerts

Evolution of Phosphorylases from N-Acetylglucosaminide Hydrolases in Family GH3

Glycoside phosphorylases hold great potential as catalysts for the synthesis of valuable sugars, glycosides, and glycans or for the development of energy-efficient microbial cell factories. However...

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases.

Carbohydrate side chain conformation is an important factor in the control of reactivity at the anomeric center, ie, in the making and breaking of glycosidic bonds, whether chemically or, for hydrolysis, by glycoside hydrolases. In nature glycosidic bond formation is catalyzed out by glycosyltransferases (GTs), glycoside phosphoryases, and transglycosidases. By analysis of 118 crystal structures of sugar nucleotide dependent (Leloir) GTs, 136 crystal structures of glycoside phosphorylases, and 54 crystal structures of transglycosidases bound to hexopyranosides or their analogs at the donor site (-1 site), we determined that most enzymes that catalyze glycoside synthesis, be they GTs, glycoside phosphorylases or transglycosidases, restrict their substrate side chains to the most reactive gauche,gauche (gg) conformation to achieve maximum stabilization of the oxocarbenium ion-like transition state for glycosyl transfer. The galactose series deviates from this trend, with α-galactosyltransferases preferentially restricting their substrates to the second-most reactive gauche,trans (gt) conformation, and β-galactosyltransferases favoring the least reactive trans,gauche (tg) conformation. This insight will help progress the design and development of improved, conformationally-restricted GT inhibitors that take advantage of these inherent side chain preferences.

Facile Enzymatic Synthesis of Diverse Naturally-Occurring β-d-Mannopyranosides Catalyzed by Glycoside Phosphorylases

β-d-Mannopyranosides are essential components of protein N-glycans, plant β-mannans, and various pathogen surface antigens. Despite their significant biological roles, the formation of 1,2-cis β-ma...

Glycoside phosphorylases for carbohydrate synthesis: An insight into the diversity and potentiality

Glycoside phosphorylases (GPs) are a major group of enzymes which catalyze the reversible formation and cleavage of glycosidic linkages, thus combining a dual functionality of both glycoside hydrolases (GHs) and glycosyltransferases (GTs). This unique advantage of reversibility, alongside with the strict regioselectivity, and the use of simple sugar phosphate donors, makes phosphorylases valuable catalysts in the synthesis of various carbohydrate structures, such as the synthesis of low-calorie sweeteners and valuable disaccharides, the synthesis of prebiotic compounds linked to health benefits, synthetic drug delivery systems and other physiological related structures, including, core components of N-linked glycans, O-linked mucin glycoproteins. Although these types of enzymes are still under-explored compared to the glycoside hydrolases and the glycosyltransferases, recently more discovered phosphorylases had been reported leading to more possibilities of synthesizing difficult glycosides. Herein we provide an overview of the distribution of GPs within the carbohydrate-active enzymes (CAZy) database, and the recent utilization of these enzymes for synthetic approaches of di- and oligosaccharides. • Glycoside phosphorylases (GPs) catalyze the reversible formation and cleavage of glycosidic linkages. • Glycoside phosphorylases have distinctive strict regioselectivity. • The reverse phosphorylsis functionality of GPs can be exploited to synthesize high value carbohydrate structures.

Short-Chain Cello-oligosaccharides: Intensification and Scale-up of Their Enzymatic Production and Selective Growth Promotion among Probiotic Bacteria.

Short-chain cello-oligosaccharides (COS; degree of polymerization, DP ≤ 6) are promising water-soluble dietary fibers. An efficient approach to their bottom-up synthesis is from sucrose and glucose using glycoside phosphorylases. Here, we show the intensification and scale up (20 mL; gram scale) of COS production to 93 g/L product and in 82 mol % yield from sucrose (0.5 M). The COS were comprised of DP 3 (33 wt %), DP 4 (34 wt %), DP 5 (24 wt %), and DP 6 (9 wt %) and involved minimal loss (≤10 mol %) to insoluble fractions. After isolation (≥95% purity; ≥90% yield), the COS were examined for growth promotion of probiotic strains. Benchmarked against inulin, trans-galacto-oligosaccharides, and cellobiose, COS showed up to 4.1-fold stimulation of cell density for Clostridium butyricum, Lactococcus lactis subsp. lactis, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus but were less efficient with Bifidobacterium sp. This study shows the COS as selectively functional carbohydrates with prebiotic potential and demonstrates their efficient enzymatic production.

Three‐Enzyme Phosphorylase Cascade Immobilized on Solid Support for Biocatalytic Synthesis of Cello−oligosaccharides

AbstractEnzyme cascades are promising for multistep biocatalytic synthesis, but their effective use beyond the proof‐of‐concept stage is challenging. Strategies to recycle the individual enzymes are critical for the applicability of such cascades. Immobilization on solid support is well developed for single enzymes but remains difficult for enzyme ensembles. Here, we show a controlled co‐immobilization of three glycoside phosphorylases to establish a highly active and recyclable biocatalyst for the conversion of sucrose and glucose into soluble (short‐chain) cello−oligosaccharides. We use protein fusion with the binding module Zbasic2 to enable non‐covalent surface tethering of all enzymes according to a uniform principle and in a programmable fashion. We thus achieve loading of the phosphorylases in an activity ratio optimal for the overall conversion and for controlling the cello−oligosaccharide chain length (≤6), hence the solubility, in the reaction. We demonstrate efficient production of ∼12 g/L cello−oligosaccharides with integrated enzyme re‐use, retaining ∼85 % of the overall initial activity after five reaction cycles. This study presents a major advance toward the practical use of systems bio‐catalysis on solid support.

The structure of a GH149 β-(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide-specific GH94 glucose-β-(1 → 3)-glucose (laminaribiose) phosphorylase.

Glycoside phosphorylases (GPs) with specificity for β‐(1 → 3)‐gluco‐oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far—glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X‐ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β‐(1 → 3)‐gluco‐oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.

Development and Application of a High-Throughput Functional Metagenomic Screen for Glycoside Phosphorylases

Glycoside phosphorylases (GPs) catalyze the reversible phosphorolysis of glycosidic bonds, releasing sugar 1-phosphates. To identify a greater range of these under-appreciated enzymes, we have developed a high-throughput functional screening method based on molybdenum blue formation. In a proof-of-principle screen focused on cellulose-degrading GPs we interrogated ∼23,000 large insert (fosmid) clones sourced from microbial communities inhabiting two separate environments and identified seven novel GPs from carbohydrate active enzyme family GH94 and one from GH149. Characterization identified cellobiose phosphorylases, cellodextrin phosphorylases, laminaribiose phosphorylases, and a β-1,3-glucan phosphorylase. To demonstrate the versatility of the screening method, varying substrate combinations were used to identify GP activity from families GH13, GH65, GH112, and GH130 in addition to GH94 and GH149. These pilot screen and substrate versatility results provide a screening paradigm platform for recovering diverse GPs from uncultivated microbial communities acting on different substrates with considerable potential to unravel previously unknown degradative pathways within microbiomes.

Unraveling the subtleties of β-(1→3)-glucan phosphorylase specificity in the GH94, GH149, and GH161 glycoside hydrolase families

Glycoside phosphorylases (GPs) catalyze the phosphorolysis of glycans into the corresponding sugar 1-phosphates and shortened glycan chains. Given the diversity of natural β-(1→3)-glucans and their wide range of biotechnological applications, the identification of enzymatic tools that can act on β-(1→3)-glucooligosaccharides is an attractive area of research. GP activities acting on β-(1→3)-glucooligosaccharides have been described in bacteria, the photosynthetic excavate Euglena gracilis, and the heterokont Ochromonas spp. Previously, we characterized β-(1→3)-glucan GPs from bacteria and E. gracilis, leading to their classification in glycoside hydrolase family GH149. Here, we characterized GPs from Gram-positive bacteria and heterokont algae acting on β-(1→3)-glucooligosaccharides. We identified a phosphorylase sequence from Ochromonas spp. (OcP1) together with its orthologs from other species, leading us to propose the establishment of a new GH family, designated GH161. To establish the activity of GH161 members, we recombinantly expressed a bacterial GH161 gene sequence (PapP) from the Gram-positive bacterium Paenibacillus polymyxa ATCC 842 in Escherichia coli. We found that PapP acts on β-(1→3)-glucooligosaccharide acceptors with a degree of polymerization (DP) ≥ 2. This activity was distinct from that of characterized GH149 β-(1→3)-glucan phosphorylases, which operate on acceptors with DP ≥ 1. We also found that bacterial GH161 genes co-localize with genes encoding β-glucosidases and ATP-binding cassette transporters, highlighting a probable involvement of GH161 enzymes in carbohydrate degradation. Importantly, in some species, GH161 and GH94 genes were present in tandem, providing evidence that GPs from different CAZy families may work sequentially to degrade oligosaccharides.

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides.

Combined with chemical synthesis, the use of glycoenzyme biocatalysts has shown great synthetic potential over recent decades owing to their remarkable versatility in terms of substrates and regio- and stereoselectivity that allow structurally controlled synthesis of carbohydrates and glycoconjugates. Nonetheless, the lack of appropriate enzymatic tools with requisite properties in the natural diversity has hampered extensive exploration of enzyme-based synthetic routes to access relevant bioactive oligosaccharides, such as cell-surface glycans or prebiotics. With the remarkable progress in enzyme engineering, it has become possible to improve catalytic efficiency and physico-chemical properties of enzymes but also considerably extend the repertoire of accessible catalytic reactions and tailor novel substrate specificities. In this review, we intend to give a brief overview of the advantageous use of engineered glycoenzymes, sometimes in combination with chemical steps, for the synthesis of natural bioactive oligosaccharides or their precursors. The focus will be on examples resulting from the three main classes of glycoenzymes specialized in carbohydrate synthesis: glycosyltransferases, glycoside hydrolases and glycoside phosphorylases.

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM-1 towards Donor Substrates Glucose/Mannose 1-Phosphate by Using X-ray Crystallography and Saturation Transfer Difference NMR Spectroscopy.

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1-phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (β-d-mannopyranosyl-(1→3)-d-glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.