Biosynthesis of cyclosporin A: partial purification and properties of a multifunctional enzyme from Tolypocladium inflatum.
An enzyme fraction most probably involved in the biosynthesis of cyclosporin A was purified 540-fold from Tolypocladium inflatum. The enzyme was capable of forming covalent enzyme-substrate complexes and catalyzed the ATP-pyrophosphate exchange reactions dependent on the unmethylated constituent amino acids of cyclosporin A. Evidence was obtained that covalent binding of substrate amino acids occurred via thioester linkage. Furthermore, the N-methylation of thio-esterified valine, leucine, and glycine residues with S-adenosyl-L-methionine was demonstrated. De novo synthesis of cyclosporin A was not observed but the formation of the diketopiperazine cyclo-(D-Ala-MeLeu) from D-alanine and L-leucine under the consumption of ATP and S-adenosyl-L-methionine. This cyclodipeptide represents a partial sequence of cyclosporin A. Molecular mass determinations revealed the enzyme activity to be lying in the range of about 700 kDa.
D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D003524:Cyclosporins / Q000096:biosynthesis*; D007930:Leucine / Q000378:metabolism; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010453:Peptide Synthases / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding
D002250:Carbon Radioisotopes; D003524:Cyclosporins; D009097:Multienzyme Complexes; C048525:cyclosporin A synthetase; D010453:Peptide Synthases; D007930:Leucine