Mann S, Marquet A, Ploux O
Synthèse, Structure et Fonction de Molécules Bioactives, UMR CNRS 7613, Université Pierre et Marie Curie, boîte 182, 4, place Jussieu, 75252 Paris Cedex 05, France.
Biochem Soc Trans. 2005 Aug;33(Pt 4):802-5. doi: 10.1042/BST0330802.
Cis and trans stereoisomers of amiclenomycin, a natural L-amino acid antibiotic, have been prepared using unequivocal routes. By using 1H NMR spectroscopy, the configuration of the six-membered ring of natural amiclenomycin was shown to be cis and not trans as originally proposed. Amiclenomycin and some synthetic analogues with the cis configuration irreversibly inactivate DAPA AT (7,8-diaminopelargonic acid aminotransferase), an enzyme involved in biotin biosynthesis, by forming an aromatic PLP (pyridoxal-5'-phosphate)-inhibitor adduct that is tightly bound to the active site. The following kinetic parameters for the inactivation of Escherichia coli DAPA AT by amiclenomycin were derived: K(I)=2 microM and k(inact)=0.4 min(-1). The structure of the aromatic adduct formed upon inactivation was confirmed by UV-visible spectroscopy, X-ray crystal structure determination and MS. Because Mycobacterium tuberculosis DAPA AT is a potential drug target, this enzyme was cloned, overexpressed and purified to homogeneity for biochemical characterization.
天然L-氨基酸抗生素阿米克霉素的顺式和反式立体异构体已通过明确的路线制备出来。通过使用1H核磁共振光谱，结果表明天然阿米克霉素六元环的构型为顺式，而非最初所提出的反式。阿米克霉素及一些具有顺式构型的合成类似物通过形成紧密结合于活性位点的芳香族磷酸吡哆醛（PLP，吡哆醛-5'-磷酸）-抑制剂加合物，不可逆地使参与生物素生物合成的7,8-二氨基壬酸转氨酶（DAPA AT）失活。得出了阿米克霉素使大肠杆菌DAPA AT失活的以下动力学参数：K(I)=2 microM，k(inact)=0.4 min(-1)。通过紫外可见光谱、X射线晶体结构测定和质谱对失活时形成的芳香族加合物的结构进行了确认。由于结核分枝杆菌DAPA AT是一个潜在的药物靶点，该酶被克隆、过量表达并纯化至同质，以进行生化特性分析。
