Molecular basis of converting proteases to ligases

Peptide asparaginyl ligases (PALs) are an unusual subgroup of asparaginyl endopeptidases (AEPs). They share the same protein structure but catalyse different reactions. AEPs hydrolyse the Asx (Asn/Asp)-Xaa bond while PALs ligate them. PALs are the processing enzymes that mediate the backbone cycliza...

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Main Author: Chang, Hong Yi
Other Authors: James P Tam
Format: Final Year Project
Language:English
Published: Nanyang Technological University 2020
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Online Access:https://hdl.handle.net/10356/140892
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Institution: Nanyang Technological University
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spelling sg-ntu-dr.10356-1408922023-02-28T18:08:42Z Molecular basis of converting proteases to ligases Chang, Hong Yi James P Tam School of Biological Sciences jptam@ntu.edu.sg Science::Biological sciences Peptide asparaginyl ligases (PALs) are an unusual subgroup of asparaginyl endopeptidases (AEPs). They share the same protein structure but catalyse different reactions. AEPs hydrolyse the Asx (Asn/Asp)-Xaa bond while PALs ligate them. PALs are the processing enzymes that mediate the backbone cyclization of plant cyclic peptides, such as cyclotides. Thus, much research has been conducted to explore factors that enable PALs to catalyse such naturally rare reactions. To date, residues in two areas near the active site are proposed to dictate ligation activity and have been named ligase-activity determinants (LADs). In this study, we aim to validate the concept of LADs by using a consensus plant AEP (cAEP) sequence. Four variants of the consensus sequence designated as cAEP(-/-), (+/+), (+/-) and (-/+), were designed where the two LADs sites either correspond to AEPs, PALs or chimera of both, respectively. The variants were recombinantly expressed in bacteria and purified for activity characterisation. Results show that cAEP(-/-) displayed AEP-like pH-dependent activity. By changing two residues, cAEP(+/+) displayed PAL-like ligase activity. In summary, the catalytic reaction governed by LADs has been validated using consensus plant AEP sequences which can be used for the discovery of PALs and the engineering of AEPs to PALs. Bachelor of Science in Biological Sciences 2020-06-02T11:11:53Z 2020-06-02T11:11:53Z 2020 Final Year Project (FYP) https://hdl.handle.net/10356/140892 en application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
spellingShingle Science::Biological sciences
Chang, Hong Yi
Molecular basis of converting proteases to ligases
description Peptide asparaginyl ligases (PALs) are an unusual subgroup of asparaginyl endopeptidases (AEPs). They share the same protein structure but catalyse different reactions. AEPs hydrolyse the Asx (Asn/Asp)-Xaa bond while PALs ligate them. PALs are the processing enzymes that mediate the backbone cyclization of plant cyclic peptides, such as cyclotides. Thus, much research has been conducted to explore factors that enable PALs to catalyse such naturally rare reactions. To date, residues in two areas near the active site are proposed to dictate ligation activity and have been named ligase-activity determinants (LADs). In this study, we aim to validate the concept of LADs by using a consensus plant AEP (cAEP) sequence. Four variants of the consensus sequence designated as cAEP(-/-), (+/+), (+/-) and (-/+), were designed where the two LADs sites either correspond to AEPs, PALs or chimera of both, respectively. The variants were recombinantly expressed in bacteria and purified for activity characterisation. Results show that cAEP(-/-) displayed AEP-like pH-dependent activity. By changing two residues, cAEP(+/+) displayed PAL-like ligase activity. In summary, the catalytic reaction governed by LADs has been validated using consensus plant AEP sequences which can be used for the discovery of PALs and the engineering of AEPs to PALs.
author2 James P Tam
author_facet James P Tam
Chang, Hong Yi
format Final Year Project
author Chang, Hong Yi
author_sort Chang, Hong Yi
title Molecular basis of converting proteases to ligases
title_short Molecular basis of converting proteases to ligases
title_full Molecular basis of converting proteases to ligases
title_fullStr Molecular basis of converting proteases to ligases
title_full_unstemmed Molecular basis of converting proteases to ligases
title_sort molecular basis of converting proteases to ligases
publisher Nanyang Technological University
publishDate 2020
url https://hdl.handle.net/10356/140892
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