טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRotem Faygenboim-Ornai
SubjectStructure-Function Relationships of Bacterial
Metallo- Aminopeptidases
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Shoham Yuval
Full Thesis textFull thesis text - English Version


Abstract

Aminopeptidases catalyze the cleavage of single amino acids from the amino terminus of peptides and protein chains. These enzymes are involved in a wide range of biological processes, such as protein digestion, protein degradation and cell-cycle control. The goal of this study was to explore the structure-function relationship of two bacterial metallo-aminopeptidases from Pseudomonas aeruginosa (PA-LAP) and Bacillus subtilis (BSAP). BSAP consists of two domains, a TIM barrel catalytic domain and a protease-associated (PA) domain. In addition, based on the crystal structure of the enzyme, the carboxy terminus of the enzyme appears to be suited near the active site and potentially blocking it. To study the roles of the PA domain and the carboxy end we prepared truncated forms of the enzyme without the PA domain or the 23 amino acid in the carboxy end, in addition to specific amino acid replacements. Native BSAP (without its leader peptide) was over-expressed in E. coli and purified using cation exchange chromatography resulting in 20-40 mg of over 95% purified protein per liter culture. The truncated form of BSAP without the PA domain was not expressed in a soluble form. In an attempt to overcome the solubility problem, the construct was fused to the extracellular xylanase gene from Geobacillus stearothermophilus and the Maltose binding protein, two highly soluble proteins. None of the fused constructs improved the solubility of the truncated protein. Removal of the carboxy end of the enzyme as well as replacements of specific residues (D155A and Y158F) involved in the interaction with the tail did not affect significantly the activity. Based on the crystal structure of BSAP, Glu452 at the carboxy-terminus tail coordinates with a Zn ion and may interfere with the incoming substrate. The replacement E452N resulted in a 100-fold improvement in the specificity constant kcat/Km (9.1*103 mM-1*s-1). The melting temperature of the E452N mutant increased by ~5 °C, suggesting that the improvement in enzyme catalysis could not be attributed to higher enzyme flexibility.

The Pseudomonas aeruginosa aminopeptidase (PA-LAP) is secreted as a non-active 58 kDa pro-enzyme which undergoes an activation process. The exact activation mechanism is still under debate. Attempts to express the PA-LAP in E. coli resulted in low yields of soluble protein. Several methods were tested to improve solubility including changing the growth temperature and fusing the gene to large soluble proteins. None of the procedures was successful.  Effective soluble production of PA-LAP was carried out in P. aeruginosa strain SS1202, that harbors an expression vector with the PA-LAP gene (pPA-LAP). In this system, PA-LAP was over produced in a soluble form and the protein was purified following two steps of ammonium sulfate precipitation. Surprisingly, the molecular mass PA-LAP in solution as determined by gel filtration, suggested that the Mr of PA-LAP is 754,000 Da. Since the calculated Mr of the recombinant PA-LAP monomer is only 57,000 Da the protein appears to form an aggregate composed of about 13 units.