Structure and function of the dihydropteroate synthase from Staphylococcus aureus.
The gene encoding the dihydropteroate synthase of staphylococcus aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and X-ray crystallographic studies. The enzyme is a dimer in solution, has a steady state kinetic mechanism that suggests random binding of the two substrates and half-site reactivity. The crystal structure of apo-enzyme and a binary complex with the substrate analogue hydroxymethylpterin pyrophosphate were determined at 2.2 A and 2.4 A resolution, respectively. The enzyme belongs to the group of "TIM-barrel" proteins and crystallizes as a non-crystallographic dimer. Only one molecule of the substrate analogue bound per dimer in the crystal. Sequencing of nine sulfonamide-resistant clinical isolates has shown that as many as 14 residues could be involved in resistance development. The residues are distributed over the surface of the protein, which defies a simple interpretation of their roles in resistance. Nevertheless, the three-dimensional structure of the substrate analogue binary complex could give important insight into the molecular mechanism of this enzyme.
Hampele
I C
IC
F. Hoffmann-La Roche Ltd, Pharma Preclinical Research Department, Basel, Switzerland.
D'Arcy
A
A
Dale
G E
GE
Kostrewa
D
D
Nielsen
J
J
Oefner
C
C
Page
M G
MG
Schönfeld
H J
HJ
Stüber
D
D
Then
R L
RL
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004094:Dihydropteroate Synthase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology*; D013420:Sulfamethoxazole / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology
D011994:Recombinant Proteins; D013449:Sulfonamides; D004094:Dihydropteroate Synthase; D013420:Sulfamethoxazole