Peptide Cyclization via Engineered Asn(OH) Residues at Neutral pH
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Solution Overview
Problem
Current methods for cyclization of peptides using peptidyl asparaginyl ligases (PALs) are limited by their preference for Asn substrates over Asp substrates, resulting in low efficiency at acidic pH due to protonation of the amine nucleophile, which hampers ligation reactions.
Innovation Solution
Introduction of Nγ-hydroxy-L-asparagine (Asn(OH)) and Nγ-amino-L-asparagine (Asn(NH2)) residues, which maintain hydrogen bonding ability at neutral pH, allowing PALs to recognize and cyclize peptides efficiently by forming interactions similar to Asn, and can be converted to Asp for further modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PALs are used for cyclization at acidic pH to maintain protonated carboxylic acid groups for enzyme binding, then hydrogen bonding ability is improved, but ligation reaction efficiency deteriorates due to protonation of the amine nucleophile
Solution Approach 1:
The invention changes the pH parameter from acidic (pH 4-5) to neutral (pH 7-8) to resolve the contradiction. At neutral pH, the amine nucleophile remains deprotonated and reactive, while the engineered S1 pocket with Asn(OH) and Asp residues maintains hydrogen bonding capability without requiring acidic conditions. This parameter change enables both reliable binding and efficient ligation.
Solution Approach 2:
The invention introduces engineered residues (Asn(OH) and Asp) in the S1 pocket as intermediaries to mediate hydrogen bonding between the substrate and enzyme. These engineered residues can form hydrogen bonds at neutral pH, acting as intermediaries that maintain binding affinity without requiring acidic pH conditions, thus enabling both binding and ligation at neutral pH.
2Productivity
If PALs are used for cyclization, then cyclic peptides are generated, but substrate scope is limited due to strong preference for P1-Asn over P1-Asp substrates
Solution Approach 1:
The invention applies local quality by engineering specific residues (Asn(OH) and Asp) at the P1 position of the S1 pocket, while leaving the rest of the enzyme structure unchanged. This localized modification creates a specialized binding environment that can accommodate both Asn and Asp substrates, expanding substrate scope without compromising the overall catalytic efficiency for cyclization.
Solution Approach 2:
The engineered S1 pocket with Asn(OH) and Asp residues confers multi-functionality on the PAL enzyme, enabling it to accept both P1-Asn and P1-Asp substrates efficiently. This universal substrate recognition capability allows the same enzyme to perform cyclization on diverse peptide substrates with different P1 residues, significantly expanding the substrate scope.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient cyclization of peptides at neutral pH, enhances binding affinity and stability, and allows for the generation of cyclic peptides with improved metabolic stability and inhibitory activities, such as MMP2 inhibitors, while maintaining the ability to convert back to Asp for specific applications.
Implementation Method 1
the sidechain amide modified by an N-hydroxyl group, has the ability to form via its the sidechain gamma-CO—NH(OH) similar interactions with the enzyme's S1 pocket as Asn
Data Source
AI summary
The present invention relates to methods that employ enzymes having Asn-specific cyclase activity as a means for generating cyclic (poly)peptides from (poly)peptides that comprise a ligation motif comprising a Nγ-hydroxy-L-asparagine or Nγ-amino-L-asparagine residue. Further encompassed are the resulting cyclic (poly)peptides and the corresponding uses.