Pacholec Michelle, Tao Junhua, Walsh Christopher T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Biochemistry. 2005 Nov 15;44(45):14969-76. doi: 10.1021/bi051599o.
During the biosynthesis of the streptomycete aminocoumarin antibiotics novobiocin and the dimeric coumermycin A(1), the bicyclic coumarin scaffold is C-methylated adjacent to the phenolic oxygen. The SAM-dependent C-methyltransferases NovO and CouO have been heterologously expressed and purified from Escherichia coli and shown to act after the aminocoumarin ring has been constructed by prior action of Nov/CouHIJK. Neither C-methyltransferase works on the tyrosyl-derived S-pantetheinyl intermediates tethered to NovH or on the subsequently released free aminocoumarin. NovL ligates the aminocoumarin to prenylhydroxybenzoate to yield novobiocic acid, which is the substrate for NovO before it is O-glycosylated by NovM. In coumermycin assembly, the corresponding ligase CouL makes the bis-amide by tandem ligation of two aminocoumarins to a dicarboxypyrrole. CouO works on both the mono- and bis-amides for mono- and di-C-methylation adjacent to the phenolic hydroxyl before it is glycosylated by CouM. Thus, the specific timing of C-methylation in the aminocoumarin antibiotic pathways is established.
在链霉菌氨基香豆素抗生素新生霉素和二聚体香豆霉素A(1)的生物合成过程中，双环香豆素骨架在酚氧相邻位置发生C-甲基化。依赖S-腺苷甲硫氨酸的C-甲基转移酶NovO和CouO已在大肠杆菌中异源表达并纯化，且显示在氨基香豆素环由Nov/CouHIJK的先前作用构建完成后发挥作用。两种C-甲基转移酶都不能作用于与NovH相连的酪氨酸衍生的S-泛酰巯基乙胺中间体，也不能作用于随后释放的游离氨基香豆素。NovL将氨基香豆素与异戊烯基羟基苯甲酸连接生成新生二羧酸，这是NovO在被NovM进行O-糖基化之前的底物。在香豆霉素组装过程中，相应的连接酶CouL通过将两个氨基香豆素串联连接到一个二羧基吡咯上形成双酰胺。CouO作用于单酰胺和双酰胺，在酚羟基相邻位置进行单甲基化和双甲基化，然后被CouM进行糖基化。因此，确定了氨基香豆素抗生素途径中C-甲基化的特定时间。
