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Dr Florian Hollfelder


Mechanistic basis of functional evolution by diversification of generalist ancestor enzymes    Gene duplication followed by sequence divergence is a key mechanism in evolution to generate functional innovation and enrich the complexity of biological systems. Though duplication events are frequently observed, the ensuing steps leading to functional differentiation between gene duplicates remain obscure. Lack of this critical information has limited both our fundamental understanding of protein evolution and the success in engineering enzymes for industrial applications.   We use laboratory evolution to investigate how a catalytically promiscuous arylsulfatase from Pseudomonas aeruginosa (PAS) can be converted into a phosphonate monoester hydrolase, displaying equivalent catalytic efficiencies for four chemically distinct substrates: phosphate diesters, phosphate, phosphonate, and sulfate monoesters. We aim at respecialising this generalist towards new activities based on a number of approaches including resurrection of ancient genes, neutrally drifted libraries, making disruptive (but innovative) libraried based on insertion/deletion mutations (rather than point substitutions). We hope that the biophysical and biochemical characterization of the selected mutants provides insight into the interplay of chemical mechanism, library character, protein structure and evolutionary strategy.


  1. Babtie, A. C., Bandyopadhyay, S., Olguin, L. F. & Hollfelder, F. Efficient catalytic promiscuity for chemically distinct reactions. Angew. Chem. Int. Ed. Engl. 48, 3692–3694 (2009). 
  2. Olguin, L. F., Askew, S. E., O'Donoghue, A. C. & Hollfelder, F. Efficient catalytic promiscuity in an enzyme superfamily: an arylsulfatase shows a rate acceleration of 1013 for phosphate monoester hydrolysis. J. Am. Chem. Soc. 130, 16547–16555 (2008). 
  3. van Loo, B.; Jonas, S.; Babtie, A. C.; Benjdia, A.; Berteau, O.; Hyvönen, M.; Hollfelder, F., An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily. Proc. Natl Acad. Sci USA 2010, 107 (7), 2740-5.

Dr Florian Hollfelder

Dr Florian Hollfelder
Department of Biochemistry
Office Phone: 01223 766048