Purification and characterization of eucaryotic alanine racemase acting as key enzyme in cyclosporin biosynthesis.
A specific alanine racemase, which is a key enzyme in the biosynthesis of the undecapeptide cyclosporin A, was purified to electrophoretic homogeneity from the fungus Tolypocladium niveum. This is the first enzyme of this kind isolated from a eucaryotic organism. The enzyme catalyzes the reversible racemization of alanine and requires pyridoxal phosphate as the exclusive cofactor. Km values for L- and D-alanine were found to be 38 and 2 mM, respectively. Maximal reaction velocity was observed at 42 degrees C and pH 8.8 for the L to D direction. Molecular mass determinations of the denatured enzyme by SDS-polyacrylamide gel electrophoresis gave a value of 37 kDa, whereas gel filtration calibration studies yielded a value between 120 and 150 kDa, indicating an oligomeric native structure.
Hoffmann
K
K
Institut fur Biochemie und Molekulare Biologie der Technischen Universität Berlin. Germany.
Schneider-Scherzer
E
E
Kleinkauf
H
H
Zocher
R
R
D000411:Alanine Racemase / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D016572:Cyclosporine / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D003904:Mitosporic Fungi / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism; D010879:Piperazines / Q000378:metabolism
D009097:Multienzyme Complexes; D010879:Piperazines; C048525:cyclosporin A synthetase; D016572:Cyclosporine; D000411:Alanine Racemase; D010453:Peptide Synthases