Journal article
Specificity versus catalytic potency: The role of threonine 44 in Escherichia coli dihydrodipicolinate synthase mediated catalysis
RCJ Dobson, MA Perugini, GB Jameson, JA Gerrard
Biochimie | ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER | Published : 2009
Abstract
In plants and bacteria, the branch point of (S)-lysine biosynthesis is the condensation of (S)-aspartate-β-semialdehyde and pyruvate, a reaction catalysed by dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52). In this study, we probe the function of threonine 44 in Escherichia coli DHDPS, with respect to its role in the proton relay. Removal of the hydroxyl moiety of threonine 44, by mutation to valine, significantly attenuates activity (0.1% of wild-type) because the proton relay is broken. It was thus predicted that mutation of threonine 44 to serine would re-establish the proton relay and thus enzymatic activity. Following site-directed mutagenesis and purification to yield the DHDPS-Thr..
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Awarded by Allan Wilson Centre
Funding Acknowledgements
The authors acknowledge Mike Griffin (University of Melbourne, Australia), Antonia Miller (University of Monash, Australia), and especially Emma McMurtrie (ITX Ltd, Australia) for critical discussion. We thank Jackie Healy for impervious, yet unruffled, technical support, and Sean Devenish for (S)-ASA and inspiration. This work was funded by the Royal Society of New Zealand Marsden Fund [contract UOC303]. Funding for the protein X-ray diffraction facility was provided, in part, by The Allan Wilson Centre for Molecular Ecology and Evolution. R.C.J.D acknowledges the University of Melbourne C.R. Roper Research Fellowship for financial support.