Marshall C G, Burkart M D, Keating T A, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 2001 Sep 4;40(35):10655-63. doi: 10.1021/bi010937s.
The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit nonribosomal peptide synthetase complex, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have tested the ability of the six-domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analogues and found the beta-functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihydrobenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclization to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N(5) and N(9) positions (condensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) analogues of vibriobactin. When L-threonyl-S-pantetheine or L-threonyl-S-(N-acetyl)cysteamine was used as a small-molecule thioester analogue of the threonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step. This clean separation of condensation from cyclization validates a two-stage mechanism for threonyl, seryl, and cysteinyl heterocyclization domains in siderophore and antibiotic synthetases. Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl carrier protein (PCP)-C2, suggesting an important role for the protein scaffold component of the heterocyclization acceptor substrate. We also examined heterocyclization donor substrate specificity at the level of acyl group and protein scaffold and observed intolerance for substitution at either position.
致病性霍乱弧菌的铁螯合肽弧菌素由四亚基非核糖体肽合成酶复合物VibE、VibB、VibH和VibF组装而成，该复合物以2,3 - 二羟基苯甲酸和L - 苏氨酸作为前体，在去甲精胺支架上形成两个通过酰胺键连接的2,3 - 二羟基苯基 - （DHP -）甲基恶唑啉基。我们测试了六结构域VibF亚基（Cy - Cy - A - C - PCP - C）利用各种L - 苏氨酸类似物的能力，发现β - 官能化氨基酸丝氨酸和半胱氨酸在氨酰 - AMP形成（腺苷化或A结构域）、氨酰 - S - 酶形成（A结构域）、被2,3 - 二氢苯甲酰 - （DHB -） - S - VibB酰化（杂环化或Cy结构域）、VibF的DHP - 恶唑啉基和DHP - 噻唑啉基 - S - 酶形式的杂环化（Cy结构域）以及在N(5)和N(9)位置转移至DHB - 去甲精胺（缩合或C结构域）以生成弧菌素的双（恶唑啉基）和双（噻唑啉基）类似物等过程中可作为替代底物。当L - 苏氨酰 - S - 泛酰巯基乙胺或L - 苏氨酰 - S -（N - 乙酰）半胱胺用作苏氨酰 - S - VibF酰基酶中间体的小分子硫酯类似物时，VibF的CyCyA片段的Cy结构域生成了缩合步骤的DHB - 苏氨酰硫酯产物，但未生成杂环化步骤的甲基恶唑啉基硫酯。缩合与环化的这种清晰分离验证了铁载体和抗生素合成酶中苏氨酰、丝氨酰和半胱氨酰杂环化结构域的两阶段机制。通过为CyCyA提供底物L - 苏氨酰 - S - 肽基载体蛋白（PCP） - C2可恢复完全的杂环化活性，这表明杂环化受体底物的蛋白质支架成分具有重要作用。我们还在酰基和蛋白质支架水平上研究了杂环化供体底物特异性，观察到在任一位置进行取代均不被耐受。
