Structure of PqsD, a Pseudomonas quinolone signal biosynthetic enzyme, in complex with anthranilate.
Structure of PqsD, a Pseudomonas quinolone signal biosynthetic enzyme, in complex with anthranilate.
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DOI：
10.1021/bi9009055
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发表时间：
2009-09-15
期刊：
影响因子：
2.9
通讯作者：
Parsons, James F.
中科院分区：
文献类型：
--
作者：
Bera, Asim K.;Atanasova, Vesna;Robinson, Howard;Eisenstein, Edward;Coleman, James P.;Pesci, Everett C.;Parsons, James F.
假单胞菌喹诺酮信号分子（PQS），2-庚基-3-羟基-4-喹诺酮，是由条件致病菌铜绿假单胞菌产生的细胞间烷基喹诺酮信号分子。在铜绿假单胞菌中，烷基喹诺酮信号传导是一种非典型系统，控制许多毒力因子的表达。PQS由色氨酸途径中间体邻氨基苯甲酸合成，邻氨基苯甲酸衍生自犬尿氨酸途径或由phnAB编码的烷基喹诺酮特异性邻氨基苯甲酸合酶。邻氨基苯甲酸通过pqsABCDE操纵子和pqsH编码的酶转化为PQS。PqsA形成活化的邻氨基苯甲酰-CoA硫酯，其将邻氨基苯甲酸穿梭至PqsD活性位点，在那里其被转移至PqsD的Cys 112。在唯一的生物化学表征的反应中，然后在邻氨基苯甲酰-PqsD和丙二酰-CoA或丙二酰-ACP（第二PqsD底物）之间发生缩合，形成2，4-二羟基喹啉（DHQ）。PqsD在其他烷基喹诺酮类（如PQS）的生物合成中所起的作用尚不清楚，尽管据报道它是其生产所必需的。然而，没有证据表明DHQ是PQS的前体。在这里，我们提出了一个结构和生物物理特性的PqsD，包括几个晶体结构的酶，包括PqsD-邻氨基苯甲酸共价中间体和无活性的Cys 112 Ala活性位点突变体与邻氨基苯甲酸复合物。结构表明PqsD在结构上类似于脂肪酸和聚酮脱氢酶的FabH和查耳酮合酶家族。晶体学不对称单元含有PqsD二聚体。PqsD单体由两个几乎相同的~170个残基的αβαβα结构域组成。结构显示邻氨基苯甲酸配体的Cys 112位于蛋白质内部深处约15 μ m长通道的底部，而第二个邻氨基苯酰辅酶A分子在通往蛋白质表面的裂缝中等待。Cys 112、His 257和Asn 287形成PqsD的FabH样催化三联体。C112 A突变体是无活性的，尽管它仍然可逆地结合邻氨基苯甲酰-CoA。邻氨基苯甲酸和Cys 112之间的共价复合物清楚地阐明了PqsD催化机制的关键元素的方向，并代表了催化循环中关键点的快照。
Pseudomonas quinolone signal (PQS), 2-heptyl-3-hydroxy-4-quinolone, is an intercellular alkyl quinolone signaling molecule produced by the opportunistic pathogen Pseudomonas aeruginosa. Alkyl quinolone signaling is an atypical system that, in P. aeruginosa, controls the expression of numerous virulence factors. PQS is synthesized from the tryptophan pathway intermediate, anthranilate, which is either derived from the kynurenine pathway or from an alkyl quinolone specific anthranilate synthase encoded by phnAB. Anthranilate is converted to PQS by the enzymes encoded by the pqsABCDE operon and pqsH. PqsA forms an activated anthraniloyl-CoA thioester that shuttles anthranilate to the PqsD active site where it is transferred to Cys112 of PqsD. In the only biochemically characterized reaction, a condensation then occurs between anthraniloyl-PqsD and malonyl-CoA or malonyl-ACP, a second PqsD substrate, forming 2,4-dihydroxyquinoline (DHQ). The role PqsD plays in the biosynthesis of other alkyl quinolones, such as PQS, is unclear though it has been reported to be required for their production. No evidence however, exists that DHQ is a PQS precursor. Here we present a structural and biophysical characterization of PqsD that includes several crystal structures of the enzyme including that of the PqsD-anthranilate covalent intermediate and the inactive Cys112Ala active site mutant in complex with anthranilate. The structure reveals that PqsD is structurally similar to the FabH and chalcone synthase families of fatty acid and polyketide synthases. The crystallographic asymmetric unit contains a PqsD dimer. The PqsD monomer is composed of two nearly identical ~170 residue αβαβα domains. The structures show anthranilate-liganded Cys112 is positioned deep in the protein interior at the bottom of a ~15 Å long channel while a second anthraniloyl-CoA molecule is waiting in the cleft leading to the protein surface. Cys112, His257, and Asn287 form the FabH-like catalytic triad of PqsD. The C112A mutant is inactive although it still reversibly binds anthraniloyl-CoA. The covalent complex between anthranilate and Cys112 clearly illuminates the orientation of key elements of the PqsD catalytic machinery and represents a snapshot of a key point in the catalytic cycle.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.2
作者：
Coleman, James P.;Hudson, L. Lynn;Pesci, Everett C.
通讯作者：
Pesci, Everett C.
影响因子：
5.7
作者：
Davies, C;Heath, RJ;Rock, CO
通讯作者：
Rock, CO
影响因子：
4.8
作者：
Jez, JM;Noel, JP
通讯作者：
Noel, JP
DOI：
10.1107/s090744499900935x
发表时间：
1999-10-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Pflugrath, JW
通讯作者：
Pflugrath, JW