Enzymatic biosynthesis of cyclosporin A and analogues

Abstract

The final assembly of the undecapeptide chain of cyclosporin A and its cycllization is accomplished in Beauveria nivea by cyclosporin synthase. This multienzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately 1 400 kDa by two different methods: 1), by 3% SDS-PAGE using the related multienzymes ACV synthetase and gramicidin S synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsCl density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of [ N- methyl-(+)-2- amino-3- hydroxy-4,4- dimethyloctanoic acid 1] CyA, dihydro-CyA, [ l-norvaline 2,5, N- methyl- lnorvaline 11] CyA, [ l-allo-isoleucine 5, N- methyl- l-allo-isoleucine 11] [ d-2-aminobutyric acid 8] CyA, [β- chloro- dalanine 8] CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [ l-threonine 2, l-leucine 5,10, d-2-hydroxyisovaleric acid 8]CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass spectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are immunosuppressive.