Miller D A, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 2001 May 1;40(17):5313-21. doi: 10.1021/bi002905v.
The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosomal peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three carrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domains (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that get heterocyclized by the Cy domains to yield a tricyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) chain lodged in thioester linkage to the PCP2 domain. The interdomain recognition by the Cy1 and Cy2 domains for the three carrier proteins was tested using inactivating mutations at the conserved serine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, and S1977A). These mutant forms of HMWP2 were tested for in trans complementation by carrier protein fragments: holo-ArCPs (S52A), holo-PCP1 and analogues (S1439A), and holo-PCP2 and analogues (S1977A). The S52A mutant tests the recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S1439A mutant tests the specificity of the same Cy1 domain for downstream substrates presented by distinct PCPs. The S1439A likewise tests the recognition of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant analogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cases in trans complementation was successful with the carrier protein fragments, allowing kinetic probes of catalytic efficiency for PCP scaffolds and for uncoupling of the condensation and heterocyclization functions of Cy1 and Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acceptor protein. This work points to the importance of protein-protein interactions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic efficiency.
鼠疫耶尔森菌素（Ybt）合成酶的HMWP2亚基是一种230 kDa的非核糖体肽合成酶（NRPS），负责合成鼠疫耶尔森菌Ybt铁载体的N端，它有一个半胱氨酸特异性腺苷化（A）结构域、三个载体蛋白结构域（ArCP、PCP1、PCP2）和两个杂环化结构域（Cy1、Cy2）。A结构域将半胱氨酸加载到两个PCP结构域上，这些半胱氨酸被Cy结构域杂环化，生成一个三环羟基苯基噻唑啉基噻唑啉基（HPTT）链，该链以硫酯键连接到PCP2结构域上。利用每个载体结构域中被磷酸泛酰巯基乙胺化的保守丝氨酸处的失活突变（S52A、S1439A和S1977A），测试了Cy1和Cy2结构域对三种载体蛋白的结构域间识别。通过载体蛋白片段对这些HMWP2的突变形式进行反式互补测试：全ArCPs（S52A）、全PCP1及其类似物（S1439A），以及全PCP2及其类似物（S1977A）。S52A突变体测试Cy1结构域对供体酰基-ArCP底物的识别，而S1439A突变体测试同一Cy1结构域对不同PCP呈现的下游底物的特异性。S1439A同样测试Cy2对其上游PCP连接的酰基供体的识别。S1977A突变体类似地测试Cy2结构域对下游Cys-PCP的识别。在所有情况下，载体蛋白片段的反式互补均成功，从而能够对PCP支架的催化效率以及Cy1和Cy2的缩合和杂环化功能的解偶联进行动力学探测。总体而言，所测试的Cy结构域对上游蛋白支架表现出明确的选择性，但对下游受体蛋白则更为宽松。这项工作指出了蛋白质-蛋白质相互作用在介导NRPS中定向链生长的重要性，并首次系统探索了蛋白质支架如何影响催化效率。
