Introduction and background
The enzyme BCAT (cytosolic isoform BCAT1 and mitochondrial isoform BCAT2) are catalyzing the conversion of branched chain amino acids isoleucine, leucine and valine to their corresponding keto acids for further degradation. The products of this process are used as sources of energy and building blocks for new molecules in cells. Interestingly, BCAT enzymes has recently been implicated in several forms of cancers, including the severe pancreatic cancer and glioblastoma (brain cancer), as these enzymes also support the rapid growing cancer cells in particular.
While the enzymatic mechanism of the BCAT enzymes is characterized at the structural and molecular level at large, we have data suggesting that the mechanism might be more complex and coordinated beyond what is the current state of knowledge. The two enzymes are dimeric, and with two active sites, there are several possibilities for coordination and regulation of the catalytic process in each site. The enzymes are highly reversible, also producing hydrophobic amino acids from keto acids under certain conditions. The activity is also regulated by red/oks sensitive elements.
Aim of project
The aim of this project is to use structural and biochemical methods to perform an in-depth investigation of the enzymatic mechanism. Different techniques and strategies will be used to understand the molecular details of the mechanism. Among the repertoire, we will design site-specific mutants with blocked active sites to understand the coordination between the two acive sites. We will use X-ray crystallography to study structures of captured reaction intermediates, and spectroscopy to follow the reaction in real-time under different conditions, such as presence of reducing agents, different ratios of substrates and products.
Project plan and methods
The project will consist of the following tasks and methods
- Use structural data to design site-specific mutants with partially blocked active sites to be used as tools to investigate potential allosteric regulation of the activity.
- Express recombinant human BCAT isoforms (both wild-type and designed mutants) using E. coli as host and purify the enzymes using chromatographic principles. Strategies to purify site-specific mutants will have to be developed, and is a major part of the project.
- Determine 3D atomic-resolution crystallographic structures of mutant BATC enzymes.
- Measure enzymatic activity of wild-type and mutant enzymes under various conditions.
Organisation / collaboration
The project is a collaboration between group leaders Bj?rn Dalhus and Lars Eide at the Department of Medical Biochemistry, University of Oslo and Oslo University Hospital (OUS). Our laboratory is located at Rikshospitalet, where we have access to relevant technologies to complete the project. Dalhus is an experienced structural biologist, with interest in drug design, and Eide is an expert in metabolism and mitochondrial biology. Hans-Petter Hersleth will serve as the internal supervisor at IBV and as a partner for the structural studies.