title: Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase.
abstract: 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.
authors.affiliation: Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 250 Mc Elroy Hall, Stillwater, Oklahoma 74078, USA.
authors.forename: Michelle Wright
authors.initials: MW
authors.lastname: Valderas
authors.forename: Babak
authors.initials: B
authors.lastname: Andi
authors.forename: William W
authors.initials: WW
authors.lastname: Barrow
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
mesh_terms: 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
chemical_list: 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