Crystal structure and resonance Raman studies of protocatechuate 3,4-dioxygenase complexed with 3,4-dihydroxyphenylacetate
Crystal structure and resonance Raman studies of protocatechuate 3,4-dioxygenase complexed with 3,4-dihydroxyphenylacetate
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DOI：
10.1021/bi970691k
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发表时间：
1997-09-23
期刊：
影响因子：
2.9
通讯作者：
Que, L
中科院分区：
文献类型：
--
作者：
Elgren, TE;Orville, AM;Que, L
报道了恶臭假单胞菌原儿茶酸3,4-双加氧酶（3,4- pcd）与替代底物3,4-二羟基苯基乙酸酯（HPCA）结合的厌氧复合物的晶体结构，其分辨率为2.4埃，R因子精细到0.17。活性位点Fe（III）的形成。HPCA螯合复合物使内源性轴向酪氨酸Tyr447 （147 β）与铁分离，并旋转成类似于先前在厌氧3,4- pcd中观察到的另一方向。3,4-二羟基苯甲酸酯配合物（3,4- pcd）。（1997）生物化学36(2):1 - 2。HPCA的芳香环在活性位点内旋转约180度的两个取向与电子密度一致。fusum短杆菌3,4- pcd的共振拉曼光谱数据。溶液中的HPCA复合物显示500 ~ 650 cm（-1）之间的低频rR振动带以及类似于1320 cm（-1）的波段，这是HPCA的诊断。铁（III）螯合物。对HPCA在C4或C3羟基上的O-18标记明确地建立了与五元螯合环体系相关的振动耦合模式。这些数据的分析表明，Fe(III)-HPCA（O4）键比Fe(III)-HPCA（O3）键短。因此，这有利于C3酚功能占据与内源性配体Tyr408(O eta) （108 beta）相反的Fe3+配体位点的晶体结构模型。这与在厌氧3,4- pcd晶体结构中提出的PCA的结合方向基本相同。PCA配合物仅基于2\F-o\-\F-c\电子密度的直接建模，并表明这是催化所需的构象。
The crystal structure of the anaerobic complex of Pseudomonas putida protocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative substrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 Angstrom resolution and refined to an R factor of 0.17. Formation of the active site Fe(III).HPCA chelated complex causes the endogenous axial tyrosinate, Tyr447 (147 beta), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in the anaerobic 3,4-PCD.3,4-dihydroxybenzoate complex (3,4-PCD.PCA) [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. Two orientations of the aromatic ring of HPCA related by an approximate 180 degrees rotation within the active site are consistent with the electron density. Resonance Raman (rR) spectroscopic data from Brevibacterium fuscum 3,4-PCD.HPCA complex in solution reveals low frequency rR vibrational bands between 500 and 650 cm(-1) as well as a band at similar to 1320 cm(-1) which are diagnostic of a HPCA.Fe(III) chelate complex. O-18 labeling of HPCA at either the C4 or C3 hydroxyl group unambiguously establishes the vibrational coupling modes associated with the five-membered chelate ring system. Analysis of these data suggests that the Fe(III)-HPCA(O4) bond is shorter than the Fe(III)-HPCA(O3) bond. This consequently favors the model for the crystal structure in which the C3 phenolic function occupies the Fe3+ ligand site opposite the endogenous ligand Tyr408(O eta) (108 beta). This is essentially the same binding orientation as proposed for PCA in the crystal structure of the anaerobic 3,4-PCD.PCA complex based solely on direct modeling of the 2\F-o\-\F-c\ electron density and suggests that this is the conformation required for catalysis.