Active site recruitment in an ancestral, bifunctional ProFAR/PRA isomerase

• Applicant: Professor Matthias Wilmanns, Ph.D.
• Subject Area: Biochemistry
• Term: from 2004 to 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5429450

Two similar (b/a)8 barrel enzymes (HisA, TrpF) catalyse isomerisation reactions in the histidine and tryptophan biosynthesis, respectively. Some microorganisms, however, comprise only one of the two genes, and complementation experiments have suggested dual substrate specificity of the corresponding gene product PriA, matching the properties of a previously hypothesized promiscuous HisA/TrpF precursor enzyme. We are proposing to mimic the evolutionary process of enzyme diversification leading to single substrate specificity. In the first step, we will determine the bifunctional catalytic properties of PriA by steady-state kinetics, determine its X-ray structure in the absence and presence of substrate analogues, and probe the contributions of several active residues to broadened substrate specificity by site directed mutagenesis. In the second step of the project, we will separately screen for HisA-like and TrpF-like PriA single substrate variants by the application of random mutagenesis methods, using E. coli strains that are auxotroph either for histidine or tryptophan.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1170: Directed Evolution to Optimise and Understand Molecular Biocatalysts