Characterisation of dihydrodipicolinate synthase (DHDPS) from Bacillus anthracis.
Bacillus anthracis is a Gram-positive spore-forming bacterium that is the causative agent of anthrax disease. The use of anthrax as a bioweapon has increased pressure for the development of an effective treatment. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the biosynthetic pathway yielding two essential bacterial metabolites, meso-diaminopimelate (DAP) and (S)-lysine. DHDPS is therefore a potential antibiotic target, as microbes require either lysine or DAP as a component of the cell wall. This paper is the first biochemical description of DHDPS from B. anthracis. Enzyme kinetic analyses, isothermal titration calorimetry (ITC), mass spectrometry and differential scanning fluorimetry (DSF) were used to characterise B. anthracis DHDPS and compare it with the well characterised Escherichia coli enzyme. B. anthracis DHDPS exhibited different kinetic behaviour compared with E. coli DHDPS, in particular, substrate inhibition by (S)-aspartate semi-aldehyde was observed for the B. anthracis enzyme (K(si(ASA))=5.4+/-0.5 mM), but not for the E. coli enzyme. As predicted from a comparison of the X-ray crystal structures, the B. anthracis enzyme was not inhibited by lysine. The B. anthracis enzyme was thermally stabilised by the first substrate, pyruvate, to a greater extent than its E. coli counterpart, but has a weaker affinity for pyruvate based on enzyme kinetics and ITC studies. This characterisation will provide useful information for the design of inhibitors as new antibiotics targeting B. anthracis.
Domigan
L J
LJ
School of Biological Sciences, University of Canterbury, Christchurch 8020, New Zealand.
Scally
S W
SW
Fogg
M J
MJ
Hutton
C A
CA
Perugini
M A
MA
Dobson
R C J
RC
Muscroft-Taylor
A C
AC
Gerrard
J A
JA
Devenish
S R A
SR
D000494:Allosteric Regulation; D000818:Animals; D001408:Bacillus anthracis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D025461:Feedback, Physiological; D005798:Genes, Bacterial; D006801:Humans; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008024:Ligands; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D004791:Enzyme Inhibitors; D008024:Ligands; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase