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Catalytic promiscuity in a protein superfamily

Reference	BB/I004327/1
Principal Investigator / Supervisor	Professor Florian Hollfelder
Co-Investigators / Co-Supervisors	Professor Marko Hyvonen
Institution	University of Cambridge
Department	Biochemistry
Funding type	Research
Value (£)	459,467
Status	Completed
Type	Research Grant
Start date	01/08/2011
End date	31/01/2015
Duration	42 months

Abstract

We have identified an enzyme that catalyses six 'promiscuous' activities for different substrate classes in addition to its postulated native activity. with remarkable second order rate accelerations ranging from 10e7 to as high as 10e19 (Proc. Natl. Acad. Sci - in press, doi/10.1073/pnas.0903951107). We want to understand how one enzyme can be so versatile - and what the fundamental mechanistic underpinnings of this wealth of 'promiscuous' activities are - by exploring phylogenetically related members of the same superfamily (yet with different promiscuity patterns) and by directed evolution that will provide snapshots of acquisition of new functions. Detailed kinetic and structural anaylsis as well as the analysis of 'fitness landscapes' for multiple promiscuous activities will provide the quantitative basis for understanding the acquisition of new function and mechanistic specialisation and despecialisation in stages of evolution.

Summary

Understanding of enzyme catalysis remains a daunting challenge, despite intense research efforts in basic and applied research. Our understanding certainly fails the most severe test - that of making catalysts that meet the efficiency of natural enzymes. This proposal addresses the determinants of catalytic efficiency in hydrolytic enzymes, using as a test bed a superfamily of catalysts that are connected by structural homology as members of the alkaline phosphatase superfamily as well as often reciprocal and crosswise catalytic promiscuity. Starting from our recent identification of one remarkable example for multiple promiscuity and high efficiency (Proc. Natl. Acad. Sci - in press, doi/10.1073/pnas.0903951107), we propose to address the factors that determine selectivity and efficiency, by structural and kinetic analysis and by directed evolution.

Impact Summary

The availability of vast amounts of sequence data allow us to consider the process of functional differentiation not only with classicial 'linear' enzymological approaches, but provides information on large numbers of 'solutions' for a catalytic problem in the form of analogues that, as in the case of PMH, form a catalytic superfamily. The mechanistic analysis of such groups of related enzymes, for multiple activities, is a conceptually novel approach that we hope will yield a more comprehensive picture of the molecular determinants of catalysis. A new technology, in vitro compartmentalisation in emulsion droplets, is ideally suited for this analysis: it is used for selections, where precise kinetic measurements can be used to select up from well above 10e6 library members. Being a screening system, this approach can also be used to characterise entire libraries, describing them as 'fitness landscapes' (in contrast to conventional directed evolution in which usually only very few selected 'hits' are characterised). It is possible that the comprehenisve characterisation of the fitness landscapes of libraries gives insight into how the evolutionary process is optimised. This approach treats directed evolution in analogy to a systems biology problem and would build a bridge between classical 'linear' and multip-parameter approaches towards understanding how protein function is brought about.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Industrial Biotechnology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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