cyclosporin is synthesized by a nonribosomal peptide synthetase, cyclosporin synthetase. the enzyme contains an adenylation domain, a thiolation domain, a condensation domain, and an n-methyltransferase domain. the adenylation domain is responsible for substrate recognition and activation, whereas the thiolation domain covalently binds the adenylated amino acids to phosphopantetheine, and the condensation domain elongates the peptide chain. cyclosporin synthetase substrates include l-valine, l-leucine, l-alanine, glycine, 2-aminobutyric acid, 4-methylthreonine, and d-alanine, which is the starting amino acid in the biosynthetic process. with the adenylation domain, cyclosporin synthetase generates the acyl-adenylated amino acids, then covalently binds the amino acid to phosphopantetheine through a thioester linkage. some of the amino acid substrates become n-methylated by s-adenosyl methionine. the cyclization step releases cyclosporin from the enzyme. amino acids such as d-ala and butenyl-methyl-l-threonine (bmt) indicate cyclosporin synthetase requires the action of other enzymes. the racemization of l-ala to d-ala by alanine racemase is pyridoxal phosphate-dependent. the formation of butenyl-methyl-l-threonine is performed by a bmt polyketide synthase that uses acetate/malonate as its starting material.