Structural determinants for peptide-bond formation by asparaginyl ligases

Hemu, Xinya; Sahili, Abbas El; Hu, Side; Wong, Ka Ho; Chen, Yu; Wong, Yee Hwa; Zhang, Xiaohong; Serra, Aida; Goh, Boon Chong; Darwis, Dina; Chen, Luyang; Sze, Siu Kwan; Liu, Chuan‐Fa; Lescar, Julien

doi:10.1073/pnas.1818568116

Cited by 100 publications

(329 citation statements)

References 56 publications

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“…6). Furthermore, the MCoAEP2 gatekeeper residue is a glycine, which is strongly indicative of hydrolase activity according to functional data reported to date 15,44 . Therefore, other factors besides the abovementioned regions must contribute to the preferential activity of AEPs.…”

Section: Discussionmentioning

confidence: 67%

“…Although covalent linkage of the C-terminus of the peptide to the enzyme physically restrained its accessible volume, we found the substrate interacted with several regions previously identified as important for activity in other cyclase AEPs, including: ligaseactivity determinant 1 (LAD1) 15 , the poly-proline loop (PPL), and the marker of ligase activity (MLA) 27 , but not LAD2 (ref. 15 ) (Fig. 4a).…”

Section: Mcoaep2 Preferentially Cyclizes Native Mcoti-ii Precursorsmentioning

confidence: 78%

“…6). For example, Tam and colleagues proposed LAD1 and LAD2 as well as the gatekeeper residue 44 (located within LAD1) as crucial for AEP ligase activity 15 . Jackson et al proposed a different region: the MLA, which comprises five amino acids whose deletion correlates with preferential ligase activity 27 .…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, other factors besides the abovementioned regions must contribute to the preferential activity of AEPs. For example, Tam and coworkers recently demonstrated that the pH can alter the activity of a particular AEP such as VyAEP1 from acting as a ligase (more neutral pH) to a hydrolase (more acid pH) 15 . Our observation that AEPs previously classified as peptidases (hydrolases) can efficiently cyclize MCoTI precursors ( Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In plants, asparaginyl endopeptidases (AEPs, also known as vacuolar processing enzymes or legumains) have been shown to recognize a conserved Asn/Asp residue at the C-terminal processing site, and cyclize the cyclotide domain by transpeptidation [9][10][11] . The AEPs characterized so far from cyclic peptide-producing plants include butelase-1 from Clitoria ternatea, OaAEP1 b from Oldenlandia affinis, VyPALs from Viola yedoensis and HaAEP1 from Helianthus annuus [12][13][14][15] . However, before cyclization, the leader peptide must be removed, and the enzyme(s) involved in this step remain poorly understood.…”

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confidence: 99%

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A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

et al. 2020

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Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a k cat /K m of 620 mM −1 s −1 , making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.

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Section: Discussionmentioning

confidence: 67%

Section: Mcoaep2 Preferentially Cyclizes Native Mcoti-ii Precursorsmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

et al. 2020

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On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation

et al. 2023

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Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell‐surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b‐C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed ‘OaAEP1b‐C247A‐∆351’ could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid‐activated OaAEP1b‐C247A enzyme. This constitutively active protein can be stored for up to 2 years at −80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E. coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation.

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Protein Engineering Strategies for Improved Pharmacokinetics

Rondon

Mahri

Morales-Yánez

et al. 2021

Adv Funct Materials

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Protein therapeutics have gained momentum in recent years and become a pillar in treating many diseases and the only choice in several ailments. Protein therapeutics are highly specific, tunable, and less toxic than conventional small drug molecules. However, reaping the full benefits of therapeutic proteins in the clinics is often hindered by issues of immunogenicity and short half-life due essentially to fast renal clearance and enzymatic degradation. Advances in polymer chemistry and protein engineering allowed overcoming some of these limitations. Strategies to prolong the half-life of proteins rely on increasing their size and stability and/or fusing them to endogenous proteins (albumin, Fc fragment of antibody) to hijack physiological pathways involved in protein recycling. On the downside, these modifications might alter therapeutic proteins structure and function. Therefore, a compromise between half-life and activity is sought. This review covers half-life extension strategies using natural and synthetic polymers as well as fusion to other proteins and sheds light on genetic engineering strategies and chemical and enzymatic reactions to achieve this goal. Promising strategies and successful applications in the clinics are highlighted. DOI: . /adfm.functions that cannot be mimicked by simple chemical compounds. Since the action of proteins is highly specific, they barely interfere with normal biological processes and cause less adverse events. Protein therapeutics are frequently derived from proteins naturally produced by the body. These agents are therefore often well tolerated and poorly immunogenic. However, proteins also suffer from significant limitations. Proteins with a molecular weight below the threshold for kidney filtration ( kDa, the size of human serum albumin) are cleared from the systemic circulation within a day. Many proteins are even cleared within a few hours or a few minutes when metabolism contributes to elimination. Therefore, therapeutic proteins need to be injected to patients several times a week (e.g., erythropoietin) or even several times a day (e.g., glucagon-like peptide-or GLP-), resulting in peaks and valleys in plasma concentrations with the alternate risks of systemic side effects and suboptimal therapeutic concentrations. Moreover, frequent administration of medication causes patient discomfort and reduces quality of life. A second limitation of proteins lies in protein immunogenicity. Foreign proteins from prokaryotes or animals might present interesting therapeutic properties in humans. However, intrinsic immunogenicity of nonhuman proteins hampers their therapeutic use in the clinic because specific antibodies generated against the foreign protein neutralize its activity and result in a loss of therapeutic efficacy over time. The unwanted immune response might even cause more serious general immune effects such as anaphylaxis.Over the last three decades, protein engineering has largely demonstrated that it can provide solutions to the limitations of natural proteins. B...

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Structural determinants for peptide-bond formation by asparaginyl ligases

Cited by 100 publications

References 56 publications

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation

Protein Engineering Strategies for Improved Pharmacokinetics

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