BIOSYNTHESIS OF THE UNUSUAL AMINO-ACID (4R)-4-[(E)-2-BUTENYL]-4-METHYL-L-THREONINE OF CYCLOSPORINE-A - IDENTIFICATION OF 3(R)-HYDROXY-4(R)-METHYL-6(E)-OCTENOIC ACID AS A KEY INTERMEDIATE BY ENZYMATIC IN-VITRO SYNTHESIS AND BY IN-VIVO LABELING TECHNIQUES

The biosynthesis of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbre viation: Bmt, systematic name: 2(S)-amino-3(R)-hydroxy-4(R)-methyl-6(E )-octenoic acid) is proposed to involve two principal phases: the form ation of a polyketide backbone and a subsequent transformation process to the final product. Here we report on the identification of 3(R)-hy droxy-4(R)-methyl-6(E)-octenoic acid as the end product of the first p hase. The primary indication of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the key intermediate in the proposed biosynthetic route came from in vivo labeling studies with [1-C-13, O-18(2)]acetate, demonstra ting retention of O-18 in the 3-hydroxy group. Final identification of this intermediate in in vitro polyketide assays with enriched enzyme fractions of Tolypocladium niveum was achieved after development of hi ghly sensitive and specific detection methods and by use of synthetic reference substances. Two additional methylated in vitro products coul d be detected and characterized as 4(R)-methyl-(E,E)-2,6-octadienoic a cid and 4(R)-methyl-6(E)-octenoic acid by liquid chromatography-mass s pectrometry analysis and comparison with synthetic reference samples. Their relevance for Bmt biosynthesis is discussed. Bmt polyketide synt hase shows optimal activity at substrate concentrations of 200 muM ace tyl-CoA, 150 muM malonyl-CoA, and 200 muM S-adenosylmethionine, around pH 7 and at 35-degrees-C. Interestingly the Bmt backbone is released from the enzyme as a coenzyme A thioester, suggesting that subsequent transformation to Bmt takes place upon this activated intermediate.