Escherichia coli aminodeoxychorismate synthase: analysis of pabB mutations affecting catalysis and subunit association.
p-Aminobenzoic acid (PABA), an essential component of the vitamin folic acid, is derived from the aromatic branch-point precursor chorismate in two steps. 4-Amino-4-deoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate, and is composed of two subunits, PabA and PabB. While various experiments have suggested that PabA and PabB act as a complex, attempts to isolate the intact complex have failed. We report here the first successful copurification of PabA and PabB by gel filtration chromatography. The association of PabA and PabB is greatly enhanced by the presence of 5 mM glutamine, and by preincubation at 37 degrees C. Conversely, the association is greatly reduced at cold temperatures. We also report the isolation and characterization of both chemically induced and site-directed mutations in PabB. Mutated PabB enzymes fall into three categories according to their properties: deficiency of chorismate amination coupled with failure to associate with PabA, deficiency of chorismate amination coupled with retention of PabA association, and competency of chorismate amination with failure of PabA association.
Rayl
E A
EA
Department of Biological Sciences, University of Illinois at Chicago 60607, USA.
Green
J M
JM
Nichols
B P
BP
D000586:Amination; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D019755:Carbon-Carbon Lyases; D019731:Carbon-Nitrogen Ligases; D003080:Cold Temperature; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D005973:Glutamine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000644:Quaternary Ammonium Compounds; D005973:Glutamine; D000637:Transaminases; C000707653:aminodeoxychorismate synthase protein, E coli; C066599:aminodeoxychorismate synthase; D019755:Carbon-Carbon Lyases; D019731:Carbon-Nitrogen Ligases