Forming coumarin C-glycosides via biocatalysis: Characterization of a C-glycosyltransferase from Angelica decursiva.

A glycosyl transferase, isolated from Angelica decursiva a medical herb rich in coumarin, shows C-glycosyl transferase activity by in vitro activity assay using 5,7-dihydroxycoumarin as substrate, producing a C-glycosylated product at position C'8 along with the main product at C'6 position. Catalytic promiscuity assay shows that AdCGT also displays O- or C-glycosylation activity to other coumarins and flavonoids. When phloretin and 2,4,6-trihydroxyacetophenone were fed as substrates, AdCGT catalyzed the formation of di-C-glycosides. Therefore, AdCGT is a multifunctional glycosyltransferase with a broad substrate acceptability. This work highlights the potential of AdCGT as a catalyst for glycosylation of coumarin and reveals a new regio-selective C-glycosyltransferase, providing a basis for exploring the mechanism of coumarin glycosylation.

Molecular Epidemiological Survey of Canine Parvovirus Circulating in China from 2014 to 2019.

The global distribution of canine parvovirus (CPV-2) derived from a closely related carnivore parvovirus poses a considerable threat to the dog population. The virus is continuously undergoing genetic evolution, giving rise to several variants. To investigate the prevalence of Chinese CPV-2 strains in recent years, a total of 30 CPV-2 strains were collected from 2018 to 2021 and the VP2 gene was sequenced and analyzed. Two variants, new CPV-2a (297Ala, 426Asn) and CPV-2c (426Glu), were identified. In contrast to previous reports, the CPV-2c variant has gained an epidemiological advantage over the new CPV-2a variant in China. To compensate for the relatively small sample size, 683 Chinese CPV-2 strains identified between 2014 and 2019 were retrieved from the GenBank database and previous publications, and analyses of these strains further supported our findings, which should be considered since the CPV-2c variant has been frequently associated with immune failure in adult dogs. VP2 protein sequence analysis revealed several amino acid substitutions, including Ala5Gly, Pro13Ser, Phe267Tyr, Tyr324Ile, Gln370Arg, Thr440Ala, and Lys570Arg. Phylogenetic analysis of full-length VP2 gene indicated a close relationship between Chinese strains and other Asian strains, suggesting mutual transmission between Asian countries. Furthermore, intercontinental transmission is a cause for concern. Surprisingly, two feline panleukopenia virus (FPV) strains with the Ile101Thr mutation in the VP2 protein were identified in canine fecal samples; FPV has been considered incapable of infecting dogs. This study clarified the epidemic characteristics of Chinese CPV-2 strains detected between 2014 and 2019, offering a reference for epidemic control. In addition, the detection of FPV in canine samples may provide information for future studies on the evolution of carnivore parvoviruses.

Structural and Molecular Dynamics Analysis of Plant Serotonin N-Acetyltransferase Reveal an Acid/Base-Assisted Catalysis in Melatonin Biosynthesis.

Serotonin N-acetyltransferase (SNAT) is the key rate-limiting enzyme in melatonin biosynthesis. It mediates melatonin biosynthesis in plants by using serotonin and 5-methoxytryptamine (5-MT), but little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl-CoA (AcCoA), serotonin, and 5-MT. OsSNAT exhibits a unique enzymatically active dimeric fold not found in the known structures of arylalkylamine N-acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket are important in catalysis, confirmed by site-directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl-transfer reaction.

Crystal structure of a S-adenosyl-L-methionine-dependent O-methyltransferase-like enzyme from Aspergillus flavus.

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) are widely distributed among almost all organisms and often characterized with conserved Rossmann fold, TIM barrel, and D×G×G×G motif. However, some MTases show no methyltransferase activity. In the present study, the crystal structure of LepI, one MTase-like enzyme isolated from A. flavus that catalyzes pericyclic reactions, was investigated to determine its structure-function relationship. The overall structure of LepI in complex with the SAM mimic S-adenosyl-L-homocysteine (SAH) (PDB ID: 6IV7) indicated that LepI is a tetramer in solution. The residues His133, Arg197, Arg295, and Asp296 located near the active site can form hydrogen bonds with the substrate, thus participating in catalytic reactions. The binding of SAH in LepI is almost identical to that in other resolved MTases; however, the location of catalytic residues differs significantly. Phylogenetic trials suggest that LepI proteins share a common ancestor in plants and algae, which may explain the conserved SAM-binding site. However, the accelerated evolution of A. flavus has introduced both functional and structural changes in LepI. More importantly, the residue Arg295, which is unique to LepI, might be a key determinant for the altered enzymatic behavior. Collectively, the differences in the composition of catalytic residues, as well as the unique tetrameric form of LepI, define its unique enzymatic behavior. The present work provides an additional understanding of the structure-function relationship of MTases and MTase-like enzymes.

Crystal structure of Oryza sativa TDC reveals the substrate specificity for TDC-mediated melatonin biosynthesis.

Plant tryptophan decarboxylase (TDC) is a type II Pyridoxal-5'-phosphate-dependent decarboxylase (PLP_DC) that could be used as a target to genetically improve crops. However, lack of accurate structural information on plant TDC hampers the understanding of its decarboxylation mechanisms. In the present study, the crystal structures of Oryza sativa TDC (OsTDC) in its complexes with pyridoxal-5'-phosphate, tryptamine and serotonin were determined. The structures provide detailed interaction information between TDC and its substrates. The Y359 residue from the loop gate is a proton donor and forms a Lewis acid-base pair with serotonin/tryptamine, which is associated with product release. The H214 residue is responsible for PLP binding and proton transfer, and its proper interaction with Y359 is essential for OsTDC enzyme activity. The extra hydrogen bonds formed between the 5-hydroxyl group of serotonin and the backbone carboxyl groups of F104 and P105 explain the discrepancy between the catalytic activity of TDC in tryptophan and in 5-hydroxytryptophan. In addition, an evolutionary analysis revealed that type II PLP_DC originated from glutamic acid decarboxylase, potentially as an adaptive evolution of mechanism in organisms in extreme environments. This study is, to our knowledge, the first to present a detailed analysis of the crystal structure of OsTDC in these complexes. The information regarding the catalytic mechanism described here could facilitate the development of protocols to regulate melatonin levels and thereby contribute to crop improvement efforts to improve food security worldwide.