Purification of ferrichrome synthetase from Aspergillus quadricinctus and characterisation as a phosphopantetheine containing multienzyme complex.
Aspergillus quadricinctus was grown under iron limitation to induce the enzymes for ferrichrome biosynthesis. The mycelium was disintegrated by ultraturrax homogenization, and ferrichrome synthetase was purified by column chromatography on DEAE cellulose, hydroxyapatite and Bio-Gel A-5m. The enzyme was almost homogeneous in single fractions as shown in gel electrophoresis under non-denaturating conditions. By fast-protein liquid chromatography on Superose 6, the purified ferrichrome synthetase (molecular weight 9.6.10(5) dissociated partly into an enzyme complex with reduced ferrichrome synthetase activity of 8 x 10(5) Da, one acetylhydroxyornithine (AHO) activating protein of 5.5 x 10(5) Da and one glycine activating protein of 4 x 10(5) Da. After SDS treatment the AHO activating protein dissociated into subunits of 9 x 10(4) Da, while the glycine activating protein dissociated into subunits of 5 x 10(4) Da and 4 x 10(4) Da in a molar ratio of 6:1. No subunits were found after SDS treatment of the larger of the two ferrichrome synthetizing enzyme complexes. Pantetheine was detected in protein bands of defined molecular weights (4 x 10(4), 9 x 10(4) and greater than 3.4 x 10(5) after SDS polyacrylamide gel electrophoresis. Gel slices were cut out, and the growth factor activity for Lactobacillus plantarum ATCC 8014 was analyzed. The calculated content was 2 mol of pantetheine per mol of ferrichrome synthetase of 9.6 x 10(5) Da.
Siegmund
K D
KD
Institut für Mikrobiologie, Universität Hannover, F.R.G.
Plattner
H J
HJ
Diekmann
H
H
D001230:Aspergillus / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D009097:Multienzyme Complexes / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D010204:Pantetheine / Q000032:analysis*; D010453:Peptide Synthases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation
D009097:Multienzyme Complexes; D010204:Pantetheine; D010453:Peptide Synthases; C029612:ferrichrome synthetase