title: Exploring the structure of glutamate racemase from Mycobacterium tuberculosis as a template for anti-mycobacterial drug discovery.
abstract: 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 Å and 1.8 Å 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 KD 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 (kcat=0.056-0.160 min(-1) and KM=0.26-0.51 mM) and can be utilized to screen proposed antimicrobial candidates for inhibition.
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Sinothai
authors.initials: S
authors.lastname: Poen
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Nakatani
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Helen K
authors.initials: HK
authors.lastname: Opel-Reading
authors.affiliation: Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand.
authors.forename: Moritz
authors.initials: M
authors.lastname: Lassé
authors.affiliation: Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Parkville, VIC 3010, Australia.
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand kurt.krause@otago.ac.nz.
authors.forename: Kurt L
authors.initials: KL
authors.lastname: Krause
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry*; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D000072417:Protein Domains
chemical_list: D001426:Bacterial Proteins; D018698:Glutamic Acid; D000591:Amino Acid Isomerases; C062292:glutamate racemase
keywords: Mycobacterium tuberculosis; analytical ultracentrifugation; enzyme kinetics; glutamate racemase; monomer engineering; thermofluor