Combining an Enhanced Polyphosphate Kinase-Driven UDP-Glucose Regeneration System with the Screening of Key Glycosyltransferases for Efficient In Vitro Synthesis of Nucleoside Disaccharides.

Nucleoside disaccharides are essential glycosides that naturally occur in specific living organisms. This study developed an enhanced UDP-glucose regeneration system to facilitate the in vitro multienzyme synthesis of nucleoside disaccharides by integrating it with nucleoside-specific glycosyltransferases. The system utilizes maltodextrin and polyphosphate as cost-effective substrates for UDP-glucose supply, catalyzed by α-glucan phosphorylase (αGP) and UDP-glucose pyrophosphorylase (UGP). To address the low activity of known polyphosphate kinases (PPKs) in the UDP phosphorylation reaction, a sequence-driven screening identified RhPPK with high activity against UDP (>1000 U/mg). Computational design further led to the creation of a double mutant with a 2566-fold increase in thermostability at 50 °C. The enhanced UDP-glucose regeneration system increased the production rate of nucleoside disaccharide synthesis by 25-fold. In addition, our UDP-glucose regeneration system is expected to be applied to other glycosyl transfer reactions.

Insights from Structure-Based Simulations into the Persulfidation of Uridine Diphosphate-Glycosyltransferase71c5 Facilitating the Reversible Inactivation of Abscisic Acid.

The action of abscisic acid (ABA) is closely related to its level in plant tissues. Uridine diphosphate-glycosyltransferase71c5 (UGT71C5) was characterized as a major UGT enzyme to catalyze the formation of the ABA-glucose ester (ABA-GE), a reversible inactive form of free ABA in Arabidopsis thaliana (thale cress). UGTs function in a mode where the catalytic base deprotonates an acceptor to allow a nucleophilic attack at the anomeric center of the donor, achieving the transfer of a glucose moiety. The proteomic data revealed that UGT71C5 can be persulfidated. Herein, an experimental method was employed to detect the persulfidation site of UGT71C5, and the computational methods were further used to identify the yet unknown molecular basis of ABA glycosylation as well as the regulatory role of persulfidation in this process. Our results suggest that the linker and the U-shaped loop are regulatory structural elements: the linker is associated with the binding of uridine diphosphate glucose (UPG) and the U-shaped loop is involved in binding both UPG and ABA.It was also found that it is through tuning the dynamics of the U-shaped loop that is accompanied by the movement of tyrosine (Y388) that the persulfidation of cysteine (C311) leads to the catalytic residue histidine (H16) being in place, preparing for the deprotonation of ABA, and then reorientates UPG and deprotonated ABA closer to the 'Michaelis' complex, facilitating the transfer of a glucose moiety. Ultimately, the persulfidation of UGT71C5 is in favor of ABA glycosylation. Our results provide insights into the molecular details of UGT71C5 recognizing substrates and insights concerning persulfidation as a possible mechanism for hydrogen sulfide (H2S) to modulate the content of ABA, which helps us understand how modulating ABA level strengthens plant tolerance.

Directional co-immobilization of artificial multimeric-enzyme complexes as a robust biocatalyst for biosynthesis curcumin glucosides and regeneration of UDP-glucose.

Site-directed protein immobilization allows the homogeneous orientation of proteins while maintaining high activity, which is advantageous for various applications. In this study, the use of SpyCatcher/SpyTag technology and magnetic nickel ferrite (NiFe2O4 NPs) nanoparticles were used to prepare a site-directed immobilization of BsUGT2m from Bacillus subtilis and AtSUSm from Arabidopsis thaliana for enhancing curcumin glucoside production with UDP-glucose regeneration from sucrose and UDP. The immobilization of self-assembled multienzyme complex (MESAs) enzymes were characterized for immobilization parameters and stability, including thermal, pH, storage stability, and reusability. The immobilized MESAs exhibited a 2.5-fold reduction in UDP consumption, enhancing catalytic efficiency. Moreover, the immobilized MESAs demonstrated high storage and temperature stability over 21 days at 4 °C and 25 °C, outperforming their free counterparts. Reusability assays showed that the immobilized MESAs retained 78.7 % activity after 10 cycles. Utilizing fed-batch technology, the cumulative titer of curcumin 4'-O-ß-D-glucoside reached 6.51 mM (3.57 g/L) and 9.45 mM (5.18 g/L) for free AtSUSm/BsUGT2m and immobilized MESAs, respectively, over 12 h. This study demonstrates the efficiency of magnetic nickel ferrite nanoparticles in co-immobilizing enzymes, enhancing biocatalysts' catalytic efficiency, reusability, and stability.

Engineering a Robust UDP-Glucose Pyrophosphorylase for Enhanced Biocatalytic Synthesis via ProteinMPNN and Ancestral Sequence Reconstruction.

UDP-glucose is a key metabolite in carbohydrate metabolism and plays a vital role in glycosyl transfer reactions. Its significance spans across the food and agricultural industries. This study focuses on UDP-glucose synthesis via multienzyme catalysis using dextrin, incorporating UTP production and ATP regeneration modules to reduce costs. To address thermal stability limitations of the key UDP-glucose pyrophosphorylase (UGP), a deep learning-based protein sequence design approach and ancestral sequence reconstruction are employed to engineer a thermally stable UGP variant. The engineered UGP variant is significantly 500-fold more thermally stable at 60 °C and has a half-life of 49.8 h compared to the wild-type enzyme. MD simulations and umbrella sampling calculations provide insights into the mechanism behind the enhanced thermal stability. Experimental validation demonstrates that the engineered UGP variant can produce 52.6 mM UDP-glucose within 6 h in an in vitro cascade reaction. This study offers practical insights for efficient UDP-glucose synthesis methods.

Molecular basis of ligand recognition specificity of flavone glucosyltransferases in Nemophila menziesii.

Of the more than 100 families of glycosyltransferases, family 1 glycosyltransferases catalyze glycosylation using uridine diphosphate (UDP)-sugar as a sugar donor and are thus referred to as UDP-sugar:glycosyl transferases. The blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin, flavone, and metal ions. Flavone 7-O-ß-glucoside-4'-O-ß-glucoside in the plant is sequentially biosynthesized from flavons by UDP-glucose:flavone 4'-O-glucosyltransferase (NmF4'GT) and UDP-glucose:flavone 4'-O-glucoside 7-O-glucosyltransferase (NmF4'G7GT). To identify the molecular mechanisms of glucosylation of flavone, the crystal structures of NmF4'G7GT in its apo form and in complex with UDP-glucose or luteolin were determined, and molecular structure prediction using AlphaFold2 was conducted for NmF4'GT. The crystal structures revealed that the size of the ligand-binding pocket and interaction environment for the glucose moiety at the pocket entrance plays a critical role in the substrate preference in NmF4'G7GT. The substrate specificity of NmF4'GT was examined by comparing its model structure with that of NmF4'G7GT. The structure of NmF4'GT may have a smaller acceptor pocket, leading to a substrate preference for non-glucosylated flavones (or flavone aglycones).

Highly Efficient Production of UDP-Glucose from Sucrose via the Semirational Engineering of Sucrose Synthase and a Cascade Route Design.

Nucleotide sugars are essential precursors for carbohydrate synthesis but are in scarce supply. Uridine diphosphate (UDP)-glucose is a core building block in nucleotide sugar preparation, making its efficient synthesis critical. Here, a process for producing valuable UDP-glucose and functional mannose from sucrose was established and improved via a semirational sucrose synthase (SuSy) design and the accurate D-mannose isomerase (MIase) cascade. Engineered SuSy exhibited enzyme activity 2.2-fold greater than that of the WT. The structural analysis identified a latch-hinge combination as the hotspot for enhancing enzyme activity. Coupling MIase, process optimization, and reaction kinetic analysis revealed that MIase addition during the high-speed UDP-glucose synthesis phase distinctly accelerated the entire process. The simultaneous triggering of enzyme modules halved the reaction time and significantly increased the UDP-glucose yield. A maximum UDP-glucose yield of 83%, space-time yield of 70 g/L/h, and mannose yield of 32% were achieved. This novel and efficient strategy for sucrose value-added exploitation has industrial promise.

Structural basis for glucosylsucrose synthesis by a member of the α-1,2-glucosyltransferase family.

Glucosylsucroses are potentially useful as additives in cosmetic and pharmaceutical formulations. Although enzymatic synthesis of glucosylsucroses is the most efficient method for their production, the key enzyme that produces them has remained unknown. Here, we report that glucosylsucrose synthase from (TeGSS) catalyzes the synthesis of glucosylsucrose using sucrose and UDP-glucose as substrates. These saccharides are homologous to glucosylsucroses produced by sp. PCC 7120 (referred to as protein alr1000). When the ratio of UDP-glucose to sucrose is relatively high, TeGSS from cyanobacteria can hydrolyze excess UDP-glucose to UDP and glucose, indicating that sucrose provides a feedback mechanism for the control of glucosylsucrose synthesis. In the present study, we solved the crystal structure of TeGSS bound to UDP and sucrose. Our structure shows that the catalytic site contains a circular region that may allow glucosylsucroses with a right-hand helical structure to enter the catalytic site. Because active site residues Tyr18 and Arg179 are proximal to UDP and sucrose, we mutate these residues (., Y18F and R179A) and show that they exhibit very low activity, supporting their role as catalytic groups. Overall, our study provides insight into the catalytic mechanism of TeGSS.

Sucrose synthase activity is not required for cellulose biosynthesis in Arabidopsis.

Biosynthesis of plant cell walls requires UDP-glucose as the substrate for cellulose biosynthesis, and as an intermediate for the synthesis of other matrix polysaccharides. The sucrose cleaving enzyme sucrose synthase (SUS) is thought to have a central role in UDP-glucose biosynthesis, and a long-held and much debated hypothesis postulates that SUS is required to supply UDP-glucose to cellulose biosynthesis. To investigate the role of SUS in cellulose biosynthesis of Arabidopsis thaliana we characterized mutants in which four or all six Arabidopsis SUS genes were disrupted. These sus mutants showed no growth phenotypes, vascular tissue cell wall defects, or changes in cellulose content. Moreover, the UDP-glucose content of rosette leaves of the sextuple sus mutants was increased by approximately 20% compared with wild type. It can thus be concluded that cellulose biosynthesis is able to employ alternative UDP-glucose biosynthesis pathway(s), and thereby the model of SUS requirements for cellulose biosynthesis in Arabidopsis can be refuted.

Catalytic flexibility of rice glycosyltransferase OsUGT91C1 for the production of palatable steviol glycosides.

Steviol glycosides are the intensely sweet components of extracts from Stevia rebaudiana. These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific ß (1-2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa, which is efficient at catalyzing ß (1-2) glucosylation. The enzyme's ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze ß (1-2) glucosylation in two distinct orientations as well as ß (1-6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable ß (1-2) glucosylation, eliminate ß (1-6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides.

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.

Biochemical Characterization of Recombinant UDPG-Dependent IAA Glucosyltransferase from Maize (Zea mays).

Here, we report a biochemical characterization of recombinant maize indole-3-acetyl-ß-d-glucose (IAGlc) synthase which glucosylates indole-3-acetic acid (IAA) and thus abolishes its auxinic activity affecting plant hormonal homeostasis. Substrate specificity analysis revealed that IAA is a preferred substrate of IAGlc synthase; however, the enzyme can also glucosylate indole-3-butyric acid and indole-3-propionic acid with the relative activity of 66% and 49.7%, respectively. KM values determined for IAA and UDP glucose are 0.8 and 0.7 mM, respectively. 2,4-Dichlorophenoxyacetic acid is a competitive inhibitor of the synthase and causes a 1.5-fold decrease in the enzyme affinity towards IAA, with the Ki value determined as 117 µM, while IAA-Asp acts as an activator of the synthase. Two sugar-phosphate compounds, ATP and glucose-1-phosphate, have a unique effect on the enzyme by acting as activators at low concentrations and showing inhibitory effect at higher concentrations (above 0.6 and 4 mM for ATP and glucose-1-phosphate, respectively). Results of molecular docking revealed that both compounds can bind to the PSPG (plant secondary product glycosyltransferase) motif of IAGlc synthase; however, there are also different potential binding sites present in the enzyme. We postulate that IAGlc synthase may contain more than one binding site for ATP and glucose-1-phosphate as reflected in its activity modulation.

31 P-MRS of the healthy human brain at 7 T detects multiple hexose derivatives of uridine diphosphate glucose.

Nucleotide sugars are required for the synthesis of glycoproteins and glycolipids, which play crucial roles in many cellular functions such as cell communication and immune responses. Uridine diphosphate-glucose (UDP-Glc) was previously believed to be the only nucleotide sugar detectable in brain by 31 P-MRS. Using spectra of high SNR and high resolution acquired at 7 T, we showed that multiple nucleotide sugars are coexistent in brain and can be measured simultaneously. In addition to UDP-Glc, these also include UDP-galactose (UDP-Gal), -N-acetyl-glucosamine (UDP-GlcNAc) and -N-acetyl-galactosamine (UDP-GalNAc), collectively denoted as UDP(G). Coexistence of these UDP(G) species is evident from a quartet-like multiplet at -9.8 ppm (M-9.8 ), which is a common feature seen across a wide age range (24-64 years). Lineshape fitting of M-9.8 allows an evaluation of all four UDP(G) components, which further aids in analysis of a mixed signal at -8.2 ppm (M-8.2 ) for deconvolution of NAD+ and NADH. For a group of seven young healthy volunteers, the concentrations of UDP(G) species were 0.04 ± 0.01 mM for UDP-Gal, 0.07 ± 0.03 mM for UDP-Glc, 0.06 ± 0.02 mM for UDP-GalNAc and 0.08 ± 0.03 mM for UDP-GlcNA, in reference to ATP (2.8 mM). The combined concentration of all UDP(G) species (average 0.26 ± 0.06 mM) was similar to the pooled concentration of NAD+ and NADH (average 0.27 ± 0.06 mM, with a NAD+ /NADH ratio of 6.7 ± 2.1), but slightly lower than previously found in an older cohort (0.31 mM). The in vivo NMR analysis of UDP-sugar composition is consistent with those from tissue extracts by other modalities in the literature. Given that glycosylation is dependent on the availability of nucleotide sugars, assaying multiple nucleotide sugars may provide valuable insights into potential aberrant glycosylation, which has been implicated in certain diseases such as cancer and Alzheimer's disease.

Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis.

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)-bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Mutation of UDP-glucose binding motif residues lead to increased affinity for ADP-glucose in sugarcane sucrose phosphate synthase.

Plant sucrose-phosphate synthase (SPS) contains a glycosyltransferase domain, which specifically catalyzes reactions with the nucleotide sugar uridine diphosphate glucose (UDP-G) as a donor substrate. Unlike plant SPS, bacterial SPS is predicted to bind other nucleotide sugars, such as adenosine diphosphate glucose (ADP-G). This study aimed to identify the UDP-G binding site of sugarcane (Saccharum officinarum) SPS (SoSPS1) and to improve its affinity for ADP-G by site-directed mutagenesis. To achieve targeted mutagenesis, amino acid distribution and comparative modeling studies were performed, followed by site-directed mutagenesis of SoSPS1 in the putative UDP-G binding motif. The N-terminal deletion of SoSPS1 (∆N-SoSPS1) was used for enzymatic analysis. The results showed that mutations in the R-X4-K, E-X7-E, and H-X5-V motifs significantly affect UDP-G and ADP-G binding. Mutations at R496 and K501 severely attenuate the affinity for UDP-G. Additionally, alanine substitutions at E591 and V570 decreased the UDP-G affinity but remarkably increased its ADP-G affinity. The R-X4-K motif plays a crucial role in the UDP-G binding site and catalytic activity of plant SPS; thus, its alteration to other amino acids was not viable. The E-X7-E and H-X5-V motifs may bind to the nucleotide glucose substrate, indicating that these motifs are involved in substrate specificity. These results agree with substrate docking simulations at the mutated residue positions, supporting the experimental results. These results demonstrate that mutation of E591 and V570 severely attenuated the UDP-G affinity, while retaining its activity against ADP-G, offering strategic insights into increasing sucrose synthesis and plant growth.

Molecular cloning and characterization of UDP-glucose: Volatile benzenoid/phenylpropanoid glucosyltransferase in petunia flowers.

Volatile benzenoids/phenylpropanoids are characteristic scent compounds in petunia flowers and are reported to be stored as glycosides in the vacuoles of petal cells. Here, we used transcriptomics and co-expression approaches with volatile benzenoid/phenylpropanoid biosynthetic genes to identify three petunia genes (UGT85A96, UGT85A97, and UGT85A98) encoding UDP-glycosyltransferase. The analyses of spatiotemporal gene expression revealed that all UGT85 genes were highly expressed in floral tissues such as petals and pistils. Functional characterization of recombinant UGT85A96 and UGT85A98 proteins expressed in Escherichia coli showed that UGT85A98 could transfer a glucosyl moiety from UDP-glucose to the hydroxyl group of various substrates including volatile benzenoids/phenylpropanoids, terpene alcohol, flavonoids, and C6 alcohol, whereas UGT85A96 specifically catalyzes the glucosylation of 2-phenylethanol and benzyl alcohol. This report describes the first experimental evidence to identify UGT enzymes that catalyze the glycosylation of volatile benzenoids/phenylpropanoids in petunia flowers.

A proposed carbon-utilization and virulence protein A, CuvA (Rv1422), from Mycobacterium tuberculosis H37Rv: crystallization, X-ray diffraction analysis and ligand binding.

Mycobacterium tuberculosis possesses the ability to undergo physiological adaptations in order to persist during the prolonged course of infection despite the active immune response of the host and in order to overcome multiple environmental changes. Previous studies have proposed that M. tuberculosis CuvA (Rv1422; MtCuvA) might play a critical role in the adaptation of the bacterium to environmental changes, such as nutrient utilization and alteration of the growth rate. However, the detailed function of MtCuvA still remains unclear owing to a lack of structural information. To better understand its role in host adaptation, MtCuvA was purified to homogeneity and was crystallized for the first time using the hanging-drop vapor-diffusion method. The crystal of MtCuvA diffracted to a resolution of 2.1 Šand belonged to the orthorhombic space group P212121, with unit-cell parameters a = 47.27, b = 170.93, c = 178.10 Å. The calculated Matthews coefficient (VM) was 2.4 Å3 Da-1, with a solvent content of 48.02%, and thus four molecules appeared to be present in the asymmetric unit. Moreover, it is reported that MtCuvA can bind to the cell-wall precursor components uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine.

Coimmobilization and colocalization of a glycosyltransferase and a sucrose synthase greatly improves the recycling of UDP-glucose: Glycosylation of resveratrol 3-O-ß-D-glucoside.

Glycosylation is one of the most efficient biocompatible methodologies to enhance the water solubility of natural products, and therefore their bioavailability. The excellent regio- and stereoselectivity of nucleotide sugar-dependent glycosyltransferases enables single-step glycosylations at specific positions of a broad variety of acceptor molecules without the requirement of protection/deprotection steps. However, the need for stoichiometric quantities of high-cost substrates, UDP-sugars, is a limiting factor for its use at an industrial scale. To overcome this challenge, here we report tailor-made coimmobilization and colocalization procedures to assemble a bi-enzymatic cascade composed of a glycosyltransferase and a sucrose synthase for the regioselective 5-O-ß-D-glycosylation of piceid with in situ cofactor regeneration. Coimmobilization and colocalization of enzymes was achieved by performing slow immobilization of both enzymes inside the porous support. The colocalization of both enzymes within the porous structure of a solid support promoted an increase in the overall stability of the bi-enzymatic system and improved 50-fold the efficiency of piceid glycosylation compared with the non-colocalized biocatalyst. Finally, piceid conversion to resveratrol 3,5-diglucoside was over 90% after 6 cycles using the optimal biocatalyst and was reused in up to 10 batch reaction cycles accumulating a TTN of 91.7 for the UDP recycling.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1.

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (ß3-ß4 loop to α0 helix) and movement of a 'shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a 'closed' state compared with its 'open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Molecular cloning and biochemical characterization of a new flavonoid glycosyltransferase from the aquatic plant lotus.

Lotus (Nelumbo nucifera Gaertn.), one of the earliest plants in angiosperms, is a perennial aquatic herb widely distributed throughout Eastern Asia. Quercetin and its glycosides are the most abundant phenolic compounds in lotus with multiple pharmacological activities. Although many flavonoid O-glycosyltransferases involved in the biosynthesis of quercetin glycosides have been identified from terrestrial plants, no glycosyltransferase has been identified in aquatic plants. In this study, a new glycosyltransferase (NpUGT6) was identified from the embryo of Nelumbo nucifera (Nelumbinis Plumula). Function characterization demonstrated that NpUGT6 exhibited a robust capability to regio- and stereo-specific O-glycosylation at the 3-hydroxy group of quercetin scaffolds with UDP-glucose. Moreover, the O-glycosylation catalyzed by NpUGT6 was reversible. NpUGT6 is the first identified flavonoid O-glycosyltransferase from aquatic plants. Its sequence will provide useful guidance for the discovery of additional flavonoid glycosyltransferses in Nymphaeaceae and other aquatic plants.

Structural dissection of sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae for substrate specificity.

Sterol glycosyltransferases catalyze the formation of a variety of glycosylated sterol derivatives and are involved in producing a plethora of bioactive natural products. To understand the molecular mechanism of sterol glycosyltransferases, we determined crystal structures of a sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae. The structures of the UGT51 and its complex with uridine diphosphate glucose (UDPG) were solved at resolutions of 2.77 Šand 1.9 Å, respectively. The structural analysis revealed that a long hydrophobic cavity, 9.2 Šin width and 17.6 Šin length located at the N-terminal domain of UGT51, is suitable for the accommodation of sterol acceptor substrates. Furthermore, a short, conserved sequence of S847-M851 was identified at the bottom of the hydrophobic cavity, which might be the steroid binding site and play an important role for the UGT51 catalytic specificity towards sterols. Molecular docking simulations indicated that changed unique interaction network in mutant M7_1 (S801A/L802A/V804A/K812A/E816K/S849A/N892D), with an 1800-fold activity improvement toward an unnatural substrate protopanaxadiol (PPD), might influence its substrate preference. This study reported the first sterol glycosyltransferase structure, providing a molecular blueprint for generating tailored sterol glycosyltransferases as potential catalytic elements in synthetic biology.