Journal article

An optimized coupled assay for quantifying diaminopimelate decarboxylase activity

Martin G Peverelli, Matthew A Perugini

BIOCHIMIE | ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER | Published : 2015

Abstract

Diaminopimelate decarboxylase (DAPDC) catalyzes the conversion of meso-DAP to lysine and carbon dioxide in the final step of the diaminopimelate (DAP) pathway in plants and bacteria. Given its absence in humans, DAPDC is a promising antibacterial target, particularly considering the rise in drug-resistant strains from pathogens such as Escherichia coli and Mycobacterium tuberculosis. Here, we report the optimization of a simple quantitative assay for measuring DAPDC catalytic activity using saccharopine dehydrogenase (SDH) as the coupling enzyme. Our results show that SDH has optimal activity at 37 °C, pH 8.0, and in Tris buffer. These conditions were subsequently employed to quantitate the ..

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University of Melbourne Researchers

Grants

Awarded by Australian Research Council


Funding Acknowledgements

We firstly would like to thank Dr. Manfred S. Weiss (Helmholtz-Zentrum Berlin) and Prof. Paul F. Cook (The University of Oklahoma) for providing the clones for M. tuberculosis DAPDC and S. cerevisiae SDH, respectively. We would also like to thank the members of the Perugini laboratory for useful discussion during the preparation of this manuscript; and funding from Australian Research Council (Future Fellowship FT0991245 and Discovery Project DP150103313) to M.A.P.