Structure and evolution of a novel dimeric enzyme from a clinically important bacterial pathogen.
Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine biosynthetic pathway. The tetrameric structure of DHDPS is thought to be essential for enzymatic activity, as isolated dimeric mutants of Escherichia coli DHDPS possess less than 2.5% that of the activity of the wild-type tetramer. It has recently been proposed that the dimeric form lacks activity due to increased dynamics. Tetramerization, by buttressing two dimers together, reduces dynamics in the dimeric unit and explains why all active bacterial DHDPS enzymes to date have been shown to be homo-tetrameric. However, in this study we demonstrate for the first time that DHDPS from methicillin-resistant Staphylococcus aureus (MRSA) exists in a monomer-dimer equilibrium in solution. Fluorescence-detected analytical ultracentrifugation was employed to show that the dimerization dissociation constant of MRSA-DHDPS is 33 nm in the absence of substrates and 29 nm in the presence of (S)-aspartate semialdehyde (ASA), but is 20-fold tighter in the presence of the substrate pyruvate (1.6 nm). The MRSA-DHDPS dimer exhibits a ping-pong kinetic mechanism (k(cat)=70+/-2 s(-1), K(m)(Pyruvate)=0.11+/-0.01 mm, and K(m)(ASA)=0.22+/-0.02 mm) and shows ASA substrate inhibition with a K(si)(ASA) of 2.7+/-0.9 mm. We also demonstrate that unlike the E. coli tetramer, the MRSA-DHDPS dimer is insensitive to lysine inhibition. The near atomic resolution (1.45 A) crystal structure confirms the dimeric quaternary structure and reveals that the dimerization interface of the MRSA enzyme is more extensive in buried surface area and noncovalent contacts than the equivalent interface in tetrameric DHDPS enzymes from other bacterial species. These data provide a detailed mechanistic insight into DHDPS catalysis and the evolution of quaternary structure of this important bacterial enzyme.
Burgess
Benjamin R
BR
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, VIC 3010, Australia.
Dobson
Renwick C J
RCJ
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, VIC 3010, Australia.
Bailey
Michael F
MF
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, VIC 3010, Australia.
Atkinson
Sarah C
SC
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, VIC 3010, Australia.
Griffin
Michael D W
MDW
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, VIC 3010, Australia.
Jameson
Geoffrey B
GB
Centre for Structural Biology, Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
Parker
Michael W
MW
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, VIC 3010, Australia; St. Vincent's Institute of Medical Research, 9 Princes St., Fitzroy, VIC 3065, Australia.
Gerrard
Juliet A
JA
School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand.
Perugini
Matthew A
MA
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, VIC 3010, Australia. Electronic address: perugini@unimelb.edu.au.
D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D019143:Evolution, Molecular; D006836:Hydro-Lyases / Q000737:chemistry*; D016106:Methicillin Resistance / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000502:physiology; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000201:enzymology*
D029968:Escherichia coli Proteins; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase