Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin 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

• Published in: The Journal of biological chemistry Vol. 268; no. 35; pp. 26127 - 26134
• Main Authors: Offenzeller, M, Su, Z, Santer, G, Moser, H, Traber, R, Memmert, K, Schneider-Scherzer, E
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15-12-1993; American Society for Biochemistry and Molecular Biology
• Subjects: Acetates - chemistry; Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid; Chromatography, Liquid; Chromatography, Thin Layer; Cyclosporine - chemistry; Fundamental and applied biological sciences. Psychology; Mass Spectrometry; Mitosporic Fungi; Proteins; Threonine - analogs & derivatives; Threonine - analysis; Threonine - biosynthesis; Fungi; Reaction intermediate; Threonine; Enzyme; Aminoacid; Biosynthesis; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)74290-2

The biosynthesis of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation: Bmt, systematic name: 2(S)-amino-3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid) is proposed to involve two principal phases: the formation of a polyketide backbone and a subsequent transformation process to the final product. Here we report on the identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the end product of the first phase. 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 [1-13C,18O2]acetate, demonstrating retention of 18O 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 highly sensitive and specific detection methods and by use of synthetic reference substances. Two additional methylated in vitro products could be detected and characterized as 4(R)-methyl-(E,E)-2,6-octadienoic acid and 4(R)-methyl-6(E)-octenoic acid by liquid chromatography-mass spectrometry analysis and comparison with synthetic reference samples. Their relevance for Bmt biosynthesis is discussed. Bmt polyketide synthase shows optimal activity at substrate concentrations of 200 microM acetyl-CoA, 150 microM malonyl-CoA, and 200 microM 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.