Active site remodelling of a cyclodipeptide synthase redefines substrate scope
Emmajay Sutherland1, Christopher John Harding1, Clarissa Melo Czekster1
1School of Biology, Biomedical Sciences Research Complex, University of St Andrews, St Andrews, Fife, UK.
Communications Chemistry
|December 15, 2022
Summary
Researchers engineered cyclodipeptide synthases (CDPSs) to create novel histidine-containing cyclic dipeptides. This study expands the chemical diversity of these bioactive molecules and offers new production methods.
Area of Science:
- Biochemistry
- Enzymology
- Medicinal Chemistry
Background:
- Cyclodipeptide synthases (CDPSs) produce cyclic dipeptides with anticancer and neuroprotective properties.
- Only two known CDPS enzymes accept histidine, limiting the diversity of histidine-containing cyclic dipeptides.
- Understanding CDPS substrate selection is crucial for discovering new enzymes and bioactive molecules.
Purpose of the Study:
- To develop an in vitro system for generating diverse histidine-containing cyclic dipeptide analogues.
- To investigate the substrate selection mechanism of a cyclo(His-Pro)-producing CDPS.
- To engineer CDPS variants with altered substrate specificity for novel cyclic dipeptide production.
Main Methods:
- Generation of an extensive library of cyclic dipeptide analogues using canonical and non-canonical amino acids.
- Determination of the crystal structure of a cyclo(His-Pro)-producing CDPS.
- Site-directed mutagenesis (single, double, and triple residue substitutions) to elucidate substrate selection and engineer enzyme variants.
Main Results:
- An in vitro system successfully expanded the chemical space of histidine-containing cyclic dipeptide analogues.
- Structural analysis and mutagenesis revealed the histidine selection mechanism within the CDPS.
- Engineered CDPS variants demonstrated altered substrate specificity, utilizing phenylalanine and leucine.
Conclusions:
- The study successfully engineered CDPS enzymes to produce novel cyclic dipeptides.
- The findings provide a foundation for directing enzyme promiscuity to create desired molecules.
- This research opens avenues for the targeted synthesis of new therapeutic cyclic dipeptides.
Related Concept Videos
01:19Allosteric Proteins-ATCase
5.8K
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
5.8K
02:53Conservative Site-specific Recombination and Phase Variation
6.1K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
6.1K
00:49Ligand Binding and Linkage
4.9K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
4.9K
01:48ATP Synthase: Mechanism
15.0K
In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased...
15.0K
01:13Induced-fit Model
81.3K
Most chemical reactions in cells require enzymes—biological catalysts that speed up the reaction without being consumed or permanently changed. They reduce the activation energy needed to convert the reactants into products. Enzymes are proteins, that usually work by binding to a substrate—a reactant molecule that they act upon.
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...
81.3K