Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains.
Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains.
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DOI：
10.1021/bi300112e
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发表时间：
2012-04-17
期刊：
影响因子：
2.9
通讯作者：
Gulick AM
中科院分区：
文献类型：
--
作者：
Mitchell CA;Shi C;Aldrich CC;Gulick AM
许多细菌使用大型模块化酶来合成聚酮化合物和肽天然产物。这些多域酶包含集成的载体域，将结合的底物传递到多个催化域，需要协调这些化学步骤。非核糖体肽合成酶 (NRPS) 通过上游腺苷酸化结构域的活性将氨基酸加载到载体结构域上。我们的实验室最近确定了包含融合腺苷酸化和载体结构域的工程化双结构域 NRPS 的结构。该结构采用域交换二聚体来说明这两个域之间的界面。为了继续我们的研究，我们现在检查 PA1221，一种来自铜绿假单胞菌的天然双结构域蛋白。我们已经确定了这种新酶的氨基酸特异性，并使用结构域特异性突变来证明在单个蛋白质分子内加载下游载体结构域比在分子间加载非融合载体结构域发生得更快。最后，我们确定了 apo- 和 Holo-PA1221 蛋白的晶体结构，后者使用缬氨酸-腺苷乙烯基磺酰胺抑制剂来捕获腺苷酸化-载体结构域相互作用。该蛋白质采用与之前的腺苷酸化载体蛋白质构建体类似的界面。将这些结构与多域 NRPS 的先前结构进行比较表明，NRPS 腺苷酸化域内的大构象变化引导载体域进入硫酯形成的活性位点。
Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Non-Ribosomal Peptide Synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a non-fused carrier domain inter-molecularly. Finally, we have determined crystal structures of both the apo- and holo-PA1221 protein, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation-carrier domain interaction. The protein adopts a similar interface to that seen with the prior adenylation-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.
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影响因子：
2.9
作者：
Horswill, AR;Escalante-Semerena, JC
通讯作者：
Escalante-Semerena, JC
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC
DOI：
10.1038/nsb810
发表时间：
2002-07-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Keating, TA;Marshall, CG;Keating, AE
通讯作者：
Keating, AE
影响因子：
--
作者：
Challis, GL;Ravel, J;Townsend, CA
通讯作者：
Townsend, CA