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Abstract: Cyclopropanation of unsaturated lipids is an intriguing enzymatic reaction and a potential therapeutic target in Mycobacterium tuberculosis. Cyclopropane fatty acid synthase from Escherichia coli is the only in vitro model available to date for mechanistic and inhibition studies. While the overall reaction mechanism of this enzymatic process is now well accepted, some mechanistic issues are still debated. Using homogeneous E. coli enzyme we have shown that, contrary to previous report based on in vivo experiments, there is no exchange of the cylopropane methylene protons with the solvent during catalysis, as probed by ultra high resolution mass spectrometry. Using [methyl-14C]-labeled and [methyl- 3H3]-S-adenosyl-l-methionine we have measured a significant intermolecular primary tritium kinetic isotope effect (T V/Kapp =1.8±0.1) consistent with a partially rate determining deprotonation step. We conclude that both chemical steps of this enzymatic cyclopropanation, the methyl addition onto the double bond and the deprotonation step, are rate determining, a common situation in efficient enzymes. [ABSTRACT FROM AUTHOR]

Amiclenomycin, a natural product containing the 1-amino cyclohexa-2,5-diene moiety is an inhibitor of 7;8-diaminopelargonic acid aminotransferase, a pyridoxal phosphate (PLP) dependent enzyme involved in biotin biosynthesis. The postulated mechanism implies the aromatisation of the Schiff base formed between PLP and amiclenomycin. Aromatic adducts have been obtained by heating PLP with amiclenomycin and other related 1-amino cyclohexa-2,5-dienes. They were fully characterized by UV–visible and ESI mass spectrometry and provide standards for identification of the enzyme-derived products. [Copyright &y& Elsevier]