Structural and functional studies of cyanide-degrading nitrilases (CHT) and their dependence on pH

• Grant number: 24/02456-6
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: May 01, 2024
• End date: April 30, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andrea Balan Fernandes
• Grantee: Valeria Del Carmen Valle-Riestra Felice
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Cyanide is a toxic compound that causes poisoning through inhalation, ingestion, or skin absorption. Due to its great affinity with metals, it is widely used as leaching for gold recovery. Cyanide is a weak acid; its dissociation constant is 9.22 at 25°C. This characteristic limits its bioremediation since it must necessarily be done at a pH greater than 9 to avoid its volatilization, and most cyanidases decrease their activity above alkaline pH. Nitrilases are enzymes that manage to break the carbon-nitrogen triple bond of CN. They are characterized by having an ±-²-²-± fold and three catalytic residues: cysteine, glutamate, and lysine. The nitrilases that catalyze cyanide degradation can be divided into two types: cyanide hydratases (CHTs) and cyanide dihydratases (CynDs). The CHT group catalyzes the substrate, obtaining a formamide as a product. The CHT sequences analyzed to date show many similarities with each other with a percentage greater than 80% identity, keeping the C-terminal highly variable. On the other hand, the CynD group converts cyanide into ammonia and formic acid. They are enzymes that have spiral structures that associate to form rods of different sizes. It has been shown that as the pH of the medium increases, the oligomerization of the enzyme decreases. The main objective of this project is to compare the enzymatic activity of CHT enzymes purified and select the one with the best performance to characterize it structurally. In addition, mutant enzyme libraries will be prepared to search for variants with activity at alkaline pH. Working with enzymes capable of maintaining cyanidase activity at alkaline pH would be highly beneficial since it would allow degradation to be carried out under conditions that limit its volatilization. (AU)