Biology subjects

Hemu, X.

Publications and source records attributed to Hemu, X..

3 recordsLinked to original sources

bioRxiv2022

Consensus design and engineering of an efficient and high-yield Peptide Asparaginyl Ligase

Plant legumains are Asn/Asp-specific endopeptidases (AEPs) that have diverse functions in plants. Peptide asparaginyl ligases (PALs) are a special legumain subtype that primarily catalyze peptide bond formation rather than hydrolysis. PALs are versatile protein engineering tools but are rarely found in nature. To overcome this limitation, here we describe a two-step method to design and engineer a high-yield and efficient recombinant PAL based on commonly found AEPs. We first constructed a consensus sequence derived from 1,500 plant legumains to design the evolutionarily stable legumain conLEG that could be produced in E. coli with 20-fold higher yield relative to that for natural legumains. We then applied the LAD (ligase-activity determinant) hypothesis to exploit conserved residues in PAL substrate-binding pockets and convert conLEG into conPAL1-3. Functional studies showed that conLEG is primarily a hydrolase, whereas conPALs are ligases. Importantly, conPAL3 is a super-efficient and broadly active PAL for peptide and protein cyclization.

biochemistry↗

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↗

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↗