Reference number : GC-2017-Q1-01748, amendment number : , agreement type : G, recipient legal name : Auclair, Karine (McGill University)|Auclair, Karine (Université McGill), recipient country : CA, recipient province : nan, recipient city : Quebec|Québec, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : Enzymes are not only fascinating catalysts, but they are also the target of most drugs. Thanks to their complex 3D structure, enzymes show many interesting characteristics rarely found in chemical catalysts. 
 
Cytochrome P450 enzymes (CYPs or P450s) are ubiquitous enzymes found across all kingdoms and involved in processes as varied as drug metabolism, biosynthesis of natural products, steroids, lipids, and more. Some P450s (e.g. CYP3A4) are known to show allosteric effects and/or use a unique cooperativity mechanism involving direct interactions between multiple copies of the same or different ligands bound to the enzyme at the same time. Such a behavior is quite challenging to delineate using existing methods. In order to study the allostery and cooperativity of CYP3A4, we have developed a new bioconjugation approach and a number of biophysical methods which will be applied. We are also currently developing a kinetic method using isothermal calorimetry. 
 
The use of enzymes in synthetic chemistry is advantageous not only because it is compatible with environmentally benign conditions but also because many enzymes catalyze reactions that are inaccessible to chemical catalysts. For example, P450s are of interest to us because of their ability to catalyze selective hydroxylations at unactivated C-H bonds. Chitinases and lignolytic peroxidases are also of interest to us because they can degrade some of the toughest and most abundant substrates, chitin and lignin respectively. We propose to develop new methods to facilitate biocatalytic applications. For example, we have pioneered the use of chemical auxiliaries to control and predict the regio- and stereo-selectivity of P450-catalyzed reactions. Using an enzyme with a large promiscuous substrate binding pocket, we have recently demonstrated that constraining the active site via rational mutagenesis can dramatically shift the selectivity of the enzyme. Finally, we have gathered preliminary data demonstrating that mechanochemistry can positively affect enzymatic processes and even surpass current chemical industrial processes. We propose to apply and optimize these methods for specific processes of interest. 
 
The overall goals of this research program are to develop new tools to study enzymes and apply these in enzymology and to facilitate biocatalysis . Representative specific aims include: 
 
- Apply our biophysical methods to study aminoglycoside 3&rsquo;-phosphoryltransferase 
- Study P450 using isothemal calorimetry in kinetics mode 
- Use our new CYP3A4 bioconjugates and generate new ones to study CYP3A4 allostery and cooperativity 
- Identify new chemical auxiliaries to control the regio- and stereo-selectivities of P450s 
- Apply, optimize and investigate our mechanochemical method with new enzymatic reactions, program purpose french : Enzymes are not only fascinating catalysts, but they are also the target of most drugs. Thanks to their complex 3D structure, enzymes show many interesting characteristics rarely found in chemical catalysts. 
 
Cytochrome P450 enzymes (CYPs or P450s) are ubiquitous enzymes found across all kingdoms and involved in processes as varied as drug metabolism, biosynthesis of natural products, steroids, lipids, and more. Some P450s (e.g. CYP3A4) are known to show allosteric effects and/or use a unique cooperativity mechanism involving direct interactions between multiple copies of the same or different ligands bound to the enzyme at the same time. Such a behavior is quite challenging to delineate using existing methods. In order to study the allostery and cooperativity of CYP3A4, we have developed a new bioconjugation approach and a number of biophysical methods which will be applied. We are also currently developing a kinetic method using isothermal calorimetry. 
 
The use of enzymes in synthetic chemistry is advantageous not only because it is compatible with environmentally benign conditions but also because many enzymes catalyze reactions that are inaccessible to chemical catalysts. For example, P450s are of interest to us because of their ability to catalyze selective hydroxylations at unactivated C-H bonds. Chitinases and lignolytic peroxidases are also of interest to us because they can degrade some of the toughest and most abundant substrates, chitin and lignin respectively. We propose to develop new methods to facilitate biocatalytic applications. For example, we have pioneered the use of chemical auxiliaries to control and predict the regio- and stereo-selectivity of P450-catalyzed reactions. Using an enzyme with a large promiscuous substrate binding pocket, we have recently demonstrated that constraining the active site via rational mutagenesis can dramatically shift the selectivity of the enzyme. Finally, we have gathered preliminary data demonstrating that mechanochemistry can positively affect enzymatic processes and even surpass current chemical industrial processes. We propose to apply and optimize these methods for specific processes of interest. 
 
The overall goals of this research program are to develop new tools to study enzymes and apply these in enzymology and to facilitate biocatalysis . Representative specific aims include: 
 
- Apply our biophysical methods to study aminoglycoside 3&rsquo;-phosphoryltransferase 
- Study P450 using isothemal calorimetry in kinetics mode 
- Use our new CYP3A4 bioconjugates and generate new ones to study CYP3A4 allostery and cooperativity 
- Identify new chemical auxiliaries to control the regio- and stereo-selectivities of P450s 
- Apply, optimize and investigate our mechanochemical method with new enzymatic reactions, agreement title english : Enzyme cooperativity, biocatalysis and bioconjugation, agreement title french : Enzyme cooperativity, biocatalysis and bioconjugation, agreement number : RGPIN, agreement value : 225000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada