Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa.
The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.
D000596:Amino Acids / Q000032:analysis; D000878:Anthranilate Synthase / Q000302:isolation & purification* / Q000378:metabolism; D002918:Chymotrypsin; D046911:Macromolecular Substances; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010196:Pancreatic Elastase; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification; D014357:Trypsin
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D000878:Anthranilate Synthase