C-Glycosyltransferases (CGTs) catalyze the formation of C-glycosidic bonds for the biosynthesis of C-glycosides, but the underlying mechanism is unclear. This process improves the solubility and bioavailability of specialized metabolites, which play important roles in plant growth and development and represent rich resources for drug discovery. Here, we performed functional and structural studies of the C-glycosyltransferase UGT708C1 from buckwheat (Fagopyrum esculentum). Enzymatic analysis showed that UGT708C1 is capable of utilizing both uridine‐59 diphosphate galactose (UDP-galactose) and uridine‐59‐diphosphate glucose (UDP-glucose) as sugar donors. Our structural studies of UGT708C1 complexed with UDP-glucose and uridine‐59‐diphosphate (UDP) identified the key roles of Asp382, Gln383, Thr151, and Thr150 in recognizing the sugar moiety of the donor substrate and Phe130, Tyr102, and Phe198 in binding and stabilizing the acceptor. A systematic site-directed mutagenesis study confirmed the important roles of these residues. Further structural analysis combined with molecular dynamics simulations revealed that phloretin binds to the acceptor-binding pocket in a bent state with a precise spatial disposition and complementarity. These findings provide insights into a catalytic mechanism for C-glycosyltransferases.

Nudix hydrolases attract considerable attention for their wide range of specialized activities in all domains of life. One particular group of Nudix phosphohydrolases (DIPPs), through their metabolism of diphosphoinositol polyphosphates (PP‐InsPs), regulates the actions of these polyphosphates upon bioenergetic homeostasis. In the current study, we describe, at an atomic level, hitherto unknown properties of human DIPP1.We provide X‐ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP‐InsPs, alternative PP‐InsP stereoisomers, or non‐hydrolysable methylene bisphosphonate analogs (“PCP‐InsPs”). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V‐shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as “suspension cables.” We demonstrate functionality for a triglycine peptide within a β‐strand which represents a non‐canonical addition to the standard Nudix catalytic core structure. We describe pre‐reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time‐resolved analysis, we uncover a chronological sequence of DIPP1/product post‐reaction states, one of which may rationalize a role for InsP ₆ as an inhibitor of catalytic activity.

Structural snapshots of protein/ligand complexes are a prerequisite for gaining atomic level insight into enzymatic reaction mechanisms. An important group of enzymes has been deprived of this analytical privilege: members of the protein tyrosine phosphatase (PTP) superfamily with catalytic WPD-loops lacking the indispensable general-acid/base within a tryptophan-proline-aspartate/glutamate context. Here, we provide the ligand/enzyme crystal complexes for one such PTP outlier: Arabidopsis thaliana Plant and Fungi Atypical Dual Specificity Phosphatase 1 (AtPFA-DSP1), herein unveiled as a regioselective and efficient phosphatase towards inositol pyrophosphate (PP-InsP) signaling molecules. Although the WPD loop is missing its canonical tripeptide motif, this structural element contributes to catalysis by assisting PP-InsP delivery into the catalytic pocket, for a choreographed exchange with phosphate reaction product. Subsequently, an intramolecular proton donation by PP-InsP substrate is posited to substitute functionally for the absent aspartate/glutamate general-acid. Overall, we expand mechanistic insight into adaptability of the conserved PTP structural elements.

Kinases that synthesize inositol phosphates (IPs) and pyrophosphates (PP-IPs) control numerous biological processes in eukaryotic cells. Herein, we extend this cellular signaling repertoire to viruses. We have biochemically and structurally characterized a minimalist inositol phosphate kinase (i.e., Tv IPK) encoded by Terrestrivirus , a nucleocytoplasmic large (“giant”) DNA virus (NCLDV). We show that Tv IPK can synthesize inositol pyrophosphates from a range of scyllo - and myo -IPs, both in vitro and when expressed in yeast cells. We present multiple crystal structures of enzyme/substrate/nucleotide complexes with individual resolutions from 1.95 to 2.6 Å. We find a heart-shaped ligand binding pocket comprising an array of positively charged and flexible side chains, underlying the observed substrate diversity. A crucial arginine residue in a conserved “G-loop” orients the γ-phosphate of ATP to allow substrate pyrophosphorylation. We highlight additional conserved catalytic and architectural features in Tv IPK, and support their importance through site-directed mutagenesis. We propose that NCLDV inositol phosphate kinases may have assisted evolution of inositol pyrophosphate signaling, and we discuss the potential biogeochemical significance of Tv IPK in soil niches.

Humans develop immunoglobulin G (IgG) antibodies to the foods they consume. In the context of food allergy, allergen-specific IgG antibodies can sequentially class-switch to pathogenic IgE. However, the mechanism underlying the antigenicity of food proteins remains uncharacterized. Here, we identified convergent antibodies arising from different antibody gene rearrangements that bind to the immunodominant peanut allergen Ara h 2 and characterized allelic and junctional constraints on germline antibody specificity. Structurally, we found similar epitope-paratope interactions across multiple gene rearrangements. We demonstrate that these germline-encoded epitope-specific convergent antibodies to peanut occur commonly in the population because of the worldwide prevalence of the relevant gene rearrangements, allelic independence, and junctional malleability. As a result, serum IgG to this public epitope is prevalent among diverse cohorts of nonallergic peanut-consuming infants and peanut-allergic children and adults. This work demonstrates that IgG recognition of dietary antigens can be intrinsically programmed by the germline antibody repertoire.