Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 145, Issue -, Pages 85-93Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2018.01.003
Keywords
Antibiotic; CD spectroscopy; Class I aldolase; DAP; Enzyme kinetics; Hydroxytetrahydrodipicolinate synthase; Sedimentation
Categories
Funding
- Australian Research Council [DP150103313]
- National Health and Medical Research Council of Australia [APP1091976]
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Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7), which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V. cholerae (Vc-DHDPS) and provide comparative structural and kinetic analyses. Structural studies employing circular dichroism spectroscopy and analytical ultracentrifugation demonstrate that the recombinant enzymes are folded and exist as dimers in solution. Kinetic analyses of untagged and His-tagged Vc-DHDPS show that the enzymes are functional with specific activities of 75.6 U/mg and 112 U/mg, K-M (pyruvate) of 0.14 mM and 0.15 mM, K-M (L-aspartate-4-semialdehyde) of 0.08 mM and 0.09 mM, and k(cat) of 34 and 46 s(-1), respectively. These results demonstrate there are no significant changes in the structure and function of Vc-DHDPS upon the addition of an N-terminal His tag and, hence, the tagged recombinant product is suitable for future studies, including screening for new inhibitors as potential anti-cholera agents. Additionally, a polyclonal antibody raised against untagged Vc-DHDPS is validated for specifically detecting recombinant and native forms of the enzyme.
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