Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 2011 Jun 28;50(25):5668-79. doi: 10.1021/bi2004922. Epub 2011 Jun 2.
The fungal peptidyl alkaloids of the tryptoquialanine and fumiquinazoline families are nonribosomally assembled by annulation of the indole side chain of fumiquinazoline F (FQF) with an alaninyl or aminoisobutyryl unit by monomodular NRPS enzymes containing adenylation, thiolation, and condensation (A-T-C) domains. The Af12060 and Af12050 enzyme pair from Aspergillus fumigatus thereby converts FQF to FQA, while the homologous TqaH and TqaB enzyme pair from Penicillium aethiopicum makes the 2'-epi diastereomer of FQA, differing only in the stereochemistry of one of the C-N bonds formed in the annulation with l-Ala. To evaluate the basis for this stereochemical control, we have mixed and matched the flavoprotein oxygenases Af12060 and TqaH with the A-T-C modular enzymes Af12050 and TqaB to show that the NRPS enzymes control the stereochemical outcome. The terminal 50 kDa condensation domains of Af12050 and TqaB are solely responsible for the stereochemical control as shown both by making chimeric (e.g., A-T-C* and A*-T*-C) forms of these monomodular NRPS enzymes and by expression, purification, and assay of the excised C-domains. The Af12050 and TqaB condensation domains are thus a paired set of diastereospecific annulation catalysts that act on the fumiquinazoline F scaffold.
真菌肽生物碱的 tryptoquialanine 和 fumiquinazoline 家族是非核糖体组装的吲哚侧链 fumiquinazoline F (FQF) 与一个丙氨酰或氨异丁酰单元通过单模块 NRPS 酶含有腺苷酸化、硫酯化和缩合 (A-T-C) 域。烟曲霉 Af12060 和 Af12050 酶对将 FQF 转化为 FQA，而青霉 Af12050 酶对从 Penicillium aethiopicum 产生 FQA 的 2'-epi 差向异构体，仅在环化过程中形成的一个 C-N 键的立体化学上有所不同用 l-Ala。为了评估这种立体化学控制的基础，我们混合和匹配了黄素蛋白加氧酶 Af12060 和 TqaH 与 A-T-C 模块化酶 Af12050 和 TqaB，以表明 NRPS 酶控制立体化学结果。Af12050 和 TqaB 的末端 50 kDa 缩合结构域仅负责立体化学控制，这既通过制作这些单模块 NRPS 酶的嵌合形式（例如，A-T-C和 A-T*-C），也通过表达、纯化和检测切除的 C 结构域来证明。因此，Af12050 和 TqaB 缩合结构域是一对具有立体特异性环化催化剂，作用于 fumiquinazoline F 支架。
