Apiose is a natural pentose containing an unusual branched-chain structure. Apiosides are bioactive natural products widely present in the plant kingdom. However, little is known on the key apiosylation reaction in the biosynthetic pathways of apiosides. In this work, we discover an apiosyltransferase GuApiGT from Glycyrrhiza uralensis. GuApiGT could efficiently catalyze 2″-O-apiosylation of flavonoid glycosides, and exhibits strict selectivity towards UDP-apiose. We further solve the crystal structure of GuApiGT, determine a key sugar-binding motif (RLGSDH) through structural analysis and theoretical calculations, and obtain mutants with altered sugar selectivity through protein engineering. Moreover, we discover 121 candidate apiosyltransferase genes from Leguminosae plants, and identify the functions of 4 enzymes. Finally, we introduce GuApiGT and its upstream genes into Nicotiana benthamiana, and complete de novo biosynthesis of a series of flavonoid apiosides. This work reports an efficient phenolic apiosyltransferase, and reveals mechanisms for its sugar donor selectivity.

Glycosylation is an important post-modification reaction in plant secondary metabolism, and contributes to structural diversity of bioactive natural products. In plants, glycosylation is usually catalyzed by UDP-glycosyltransferases. Flavonoid 2′-O-glycosides are rare glycosides. However, no UGTs have been reported, thus far, to specifically catalyze 2′-O-glycosylation of flavonoids. In this work, UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-O-glycosyltransferase. It could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new compounds. Some of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2. The crystal structure of UGT71AP2 (2.15 Å) was solved, and mechanisms of its regio-selectivity was interpreted by pK_a calculations, molecular docking, MD simulation, MM/GBSA binding free energy, QM/MM, and hydrogen‒deuterium exchange mass spectrometry analysis. Through structure-guided rational design, we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity (the ratio of 7-O-glycosylation byproducts decreased from 48% to 4%) and catalytic efficiency of 2′-O-glycosylation (k_cat/K_m, 0.23 L/(s·μmol), 12-fold higher than the native). Moreover, UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity, and is a dual function glycosyltransferase. This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.

Primary supranuclear palsy (PSP) is a rare neurodegenerative disease that perturbs body movement, eye movement, and walking balance. Similar to Alzheimer's disease (AD), the abnormal aggregation of tau fibrils in the central neuronal and glial cells is a major hallmark of PSP disease. In this study, we use multiple approaches, including docking, molecular dynamics, and metadynamics simulations, to investigate the binding mechanism of 10 first- and second-generations of PET tracers for PSP tau and compare their binding in cortical basal degeneration (CBD) and AD tauopathies. Structure-activity relationships, binding preferences, the nature of ligand binding in terms of basic intermolecular interactions, the role of polar/charged residues, induced-fit mechanisms, grove closures, and folding patterns for the binding of these tracers in PSP, CBD, and AD tau fibrils are evaluated and discussed in detail in order to build a holistic picture of what is essential for the binding and also to rank the potency of the different tracers. For example, we found that the same tracer shows different binding preferences for the surface sites of tau fibrils that are intrinsically distinct in the folding patterns. Results from the metadynamics simulations predict that PMPBB3 and PBB3 exhibit the strongest binding free energies onto the Q²⁷⁶[I²⁷⁷]I²⁷⁸, Q³⁵¹[S³⁵²]K³⁵³, and N³⁶⁸[K³⁶⁹]K³⁷⁰ sites of PSP than the other explored tracers, indicating a solid preference for vdW and cation-π interactions. Our results also reproduced known preferences of tracers, namely, that MK6240 binds better to AD tau than CBD tau and PSP tau and that CBD2115, PI2620, and PMPBB3 are 4R tau binders. These findings fill in the well-sought-after knowledge gap in terms of these tracers' potential binding mechanisms and will be important for the design of highly selective novel PET tracers for tauopathies.

C-Glycosides are important natural products with various bioactivities. In plant biosynthetic pathways, the C-glycosylation step is usually catalyzed by C-glycosyltransferases (CGTs), and most of them prefer to accept uridine 5′-diphosphate glucose (UDP-Glc) as sugar donor. No CGTs favoring UDP-rhamnose (UDP-Rha) as sugar donor has been reported, thus far. Herein, we report the first selective C-rhamnosyltransferase VtCGTc from the medicinal plant Viola tricolor. VtCGTc could efficiently catalyze C-rhamnosylation of 2-hydroxynaringenin 3-C-glucoside, and exhibited high selectivity towards UDP-Rha. Mechanisms for the sugar donor selectivity of VtCGTc were investigated by molecular dynamics (MD) simulations and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) binding free energy calculations. Val144 played a vital role in recognizing UDP-Rha, and the V144T mutant could efficiently utilize UDP-Glc. This work provides a new and efficient approach to prepare flavonoid C-rhamnosides such as violanthin and iso-violanthin.

The α-synucleinopathies are a group of neurodegenerative diseases characterized by the deposition of α-synuclein aggregates (α-syn) in the brain. Currently, there is no suitable tracer to enable a definitive early diagnosis of these diseases. We reported candidates based on 4,4′-disarylbisthiazole (DABTA) scaffold with a high affinity towards α-syn and excellent selectivity over Aβ and tau fibrils. Based on prior in silico studies, a focused library of 23 halogen-containing and O-methylated DABTAs was prepared. The DABTAs were synthesized via a modified two-step Hantzsch thiazole synthesis, characterized, and used in competitive binding assays against [³H]PiB and [³H]DCVJ. The DABTAs were obtained with an overall chemical yield of 15–71%, and showed a calculated lipophilicity of 2.5–5.7. The ligands demonstrated an excellent affinity to α-syn with both [³H]PiB and [³H]DCVJ: K_i 0.1–4.9 nM and up to 20–3900-fold selectivity over Aβ and tau fibrils. It could be concluded that in silico simulation is useful for the rational design of a new generation of DABTAs. Further investigation of the leads in the next step is encouraged: radiolabeling of the ligands with radioisotopes such as fluorine-18 or carbon-11 for in vivo, ex vivo, and translational research and for further in vitro experiments on human-derived protein aggregates.