Protein Engineering Elucidates the Relationship between Structure, Function and Stability of a Metabolic Enzyme

The relationship between oligomerisation state, stability, and catalytic activity of the anthranilate phosphoribosyl transferase from Sulfolobus solfataricus (sAnPRT) was analysed by three interrelated protein engineering approaches. The extremely thermostable homodimeric sAnPRT enzyme was converted into a monomer by rational design, and its low catalytic activity at 37 C was elevated by a combination of random mutagenesis and metabolic selection in the mesophilic host Escherichia coli. The two amino acid exchanges leading to monomerization and the two substitutions resulting in activation of sAnPRT were then combined, which resulted in an ‘‘activated monomer’’ that was significantly less stable and more active than wild-type sAnPRT. Using a combination of random mutagenesis and selection in the thermophilic host Thermus thermophilus, the activated monomer was stabilized, and the consequences of stabilization for catalytic activity and association state were analysed. 85 http://www.beilstein-institut.de/escec2011/Proceedings/Sterner/Sterner.pdf Experimental Standard Conditions of Enzyme Characterization September 12 – 16, 2011, Rüdesheim/Rhein, Germany