Biology subjects

Tam, J. P.

Publications and source records attributed to Tam, J. P..

3 recordsLinked to original sources

bioRxiv2022

Substrate-binding Glycine Residues are Major Determinants for Hydrolase and Ligase Activity of Plant Legumains

Peptide asparaginyl ligases (PALs) are Asn/Asp(Asx)-specific ligases that are useful for precision modifications of proteins and live-cell surfaces. However, PALs share high structural similarity to the far more common asparaginyl endopeptidases (AEPs), also known as legumains that hydrolyze peptide bonds after Asx, thus making it challenging to identify PALs in a sea of AEPs. Previously we identified sequences flanking the catalytic site as ligase activity determinants (LADs) for legumains. Here we show that two conserved substrate-binding Gly residues are critical, but negative determinants for ligase activity, based on a combined bioinformatics analysis of 1,500 plant legumains, mutagenesis and functional study of 16 novel legumains, plus identification of seven new PALs. We also show that PALs are rare and AEPs are much more common, accounting for about 1% and 88%, respectively. Our results suggest that specific glycine residues are molecular determinants to identify PALs and AEPs as two different legumain subfamilies.

plant biology↗

bioRxiv2022

Genomic, transcriptomic, and metabolomic analysis of Traditional Chinese Medicine plant Oldenlandia corymbosa reveals the biosynthesis and mode of action of anti-cancer metabolites

Natural products from traditional medicinal plants are valuable candidates for clinical cancer therapy. Plants from the Oldenlandia-Hedyotis complex are popular ingredients of Traditional Chinese Medicine (TCM), however a major hurdle in the plant bioprospecting process of TCM plants is that the active metabolites, their biosynthetic pathways, and mode of action are often unknown. We show that Oldenlandia corymbosa extracts are active against breast cancer cell lines. To study the genes involved in the biosynthesis of active compounds in this medicinal plant, we assembled a high-quality genome. We show that the main active compound is ursolic acid and that abiotic stresses cause changes in anti-cancer activity, metabolite composition, and gene expression of plants. To reveal the mode of action of ursolic acid, we show that cancer cells undergo mitotic catastrophe, and we identify three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking.

plant biology↗

bioRxiv2021

Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis

Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzymes S1 pocket and why a hydrophobic residue is required at the substrate P2 position. Beside illustrating the role played by P1 and P2 residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the "Gatekeeper" residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities. One sentence summaryWe captured VyPAL2, a catalytically efficient plant peptide ligase with a bound substrate, providing the molecular basis for substrate recognition and ligation.

biochemistry↗