Journal
BIOCHEMICAL JOURNAL
Volume 473, Issue -, Pages 1267-1280Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BCJ20160186
Keywords
analytical ultracentrifugation; enzyme kinetics; glutamate racemase; monomer engineering; Mycobacterium tuberculosis; thermofluor
Categories
Funding
- Royal Society of New Zealand
- Thrash Foundation, Houston, Texas
- University of Otago
- Ministry of Business, Innovation and Employment [UOCX1208]
- New Zealand Royal Society Marsden Fund [UOC1013]
- U.S. Army Research Laboratory
- U.S. Army Research Office [W911NF-11-1-0481]
- New Zealand Ministry of Business, Innovation & Employment (MBIE) [UOCX1208] Funding Source: New Zealand Ministry of Business, Innovation & Employment (MBIE)
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Glutamate racemase (MurI) is responsible for providing D-glutamate for peptidoglycan biosynthesis in bacteria and has been a favoured target in pharmaceutical drug design efforts. It has recently been proven to be essential in Mycobacterium tuberculosis, the causative organism of tuberculosis, a disease for which new medications are urgently needed. In the present study, we have determined the protein crystal structures of MurI from both M. tuberculosis and Mycobacterium smegmatis in complex with D-glutamate to 2.3 angstrom and 1.8 angstrom resolution respectively. These structures are conserved, but reveal differences in their active site architecture compared with that of other MurI structures. Furthermore, compounds designed to target other glutamate racemases have been screened but do not inhibit mycobacterial MurI, suggesting that a new drug design effort will be needed to develop inhibitors. A new type of MurI dimer arrangement has been observed in both structures, and this arrangement becomes the third biological dimer geometry for MurI found to date. The mycobacterial MurI dimer is tightly associated, with a K-D in the nanomolar range. The enzyme binds D- and L-glutamate specifically, but is inactive in solution unless the dimer interface is mutated. We created triple mutants of this interface in the M. smegmatis glutamate racemase (D26R/R105A/G194R or E) that have appreciable activity (k(cat) = 0.056-0.160 min(-1) and K-M = 0.26-0.51 mM) and can be utilized to screen proposed antimicrobial candidates for inhibition.
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