SUBSTRATE SPECIFICITIES OF CYCLOSPORINE SYNTHETASE AND PEPTOLIDE SDZ 214-103 SYNTHETASE - COMPARISON OF THE SUBSTRATE SPECIFICITIES OF THE RELATED MULTIFUNCTIONAL POLYPEPTIDES

The recently discovered multifunctional polypeptide cyclosporin synthe tase is capable of synthesizing the cyclic undecapeptide cyclosporin A in a batch reaction. Substrates are the unmethylated constitutive ami no acids of cyclosporin A. Exchange of one or more of these by various amino acids gives a picture of the substrate specificity of the enzym e in vitro, which is different from the known picture obtained by in v ivo studies. The uncommon amino acid butenylmethyl-threonine in positi on 1 of the cyclosporin ring can be exchanged by an unexpected large s pectrum of different amino acids, showing a great flexibility of this site. Position 2, on the other hand, which shows the greatest variabil ity in vivo, has an only slightly lower specificity in vitro. Position 3 has a very high degree of specificity; positions 4, 6, 7, 9, and 10 have marginally less. The variability of positions 5 and 11 is modera te, whereas position 8 shows only low substrate specificity in vitro. In general, most sites of SDZ 214-103 synthetase appear to be more spe cific than those of cyclosporin synthetase. Site 11 has nearly identic al substrate specificity compared with that of cyclosporin synthetase. The D-2-hydroxy acid position (position 8) can be occupied by a large spectrum of substrates varying from D-lactic acid to D-2-hydroxyisoca proic acid. Within the limits of the present data, the addition of fur ther functional groups to the D-2-hydroxy acid moiety are apparently n ot tolerated by the enzyme.