Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase.
Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.
Valderas
Michelle Wright
MW
Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 250 Mc Elroy Hall, Stillwater, Oklahoma 74078, USA.
Andi
Babak
B
Barrow
William W
WW
Cook
Paul F
PF
D010129:4-Aminobenzoic Acid / Q000737:chemistry / Q000378:metabolism; D001408:Bacillus anthracis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004094:Dihydropteroate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000378:metabolism; D005636:Fructosephosphates / Q000737:chemistry / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017616:Magnesium Compounds / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010756:Phosphoric Acids / Q000737:chemistry / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D011622:Pterins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014305:Triose-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism
C492819:6-hydroxymethyl-7,8-dihydropterin pyrophosphate; D011756:Diphosphates; D005636:Fructosephosphates; D017616:Magnesium Compounds; D010756:Phosphoric Acids; D011622:Pterins; D011994:Recombinant Proteins; D013449:Sulfonamides; D009243:NAD; C027618:fructose-6-phosphate; C034732:magnesium pyrophosphate; D005993:Glycerolphosphate Dehydrogenase; D004094:Dihydropteroate Synthase; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; D005634:Fructose-Bisphosphate Aldolase; D014305:Triose-Phosphate Isomerase; D010129:4-Aminobenzoic Acid