The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate:: Insights into its biological function
The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate:: Insights into its biological function
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DOI：
10.1110/ps.072801907
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发表时间：
2007-07-01
期刊：
影响因子：
8
通讯作者：
Rayment, Ivan
中科院分区：
文献类型：
--
作者：
Garvey, Graeme S.;Rocco, Christopher J.;Rayment, Ivan
在细菌中，在2-甲基柠檬酸循环中，2-甲基柠檬酸脱水生成2-甲基乌头酸是由无辅因子（PrpD）酶或乌头酸酶样（AcnD）酶催化的。使用AcnD的细菌也需要PrpF蛋白的功能，其功能以前是未知的。为了深入了解PrpF的功能，在2.0埃分辨率下解析了来自细菌Shewanella oneidensis的PrpF蛋白的三维晶体结构。PrpF的蛋白质折叠与流感嗜血杆菌的非PLP依赖性二氨基庚二酸差向异构酶（一种来自羊种布鲁氏菌的推定脯氨酸消旋酶）以及最近沉积的来自铜绿假单胞菌的假定蛋白质的结构惊人相似。体外研究结果表明，PrpF异构化反乌头酸顺乌头酸。有人提出，PrpF催化的顺-反异构化的收益通过碱催化质子提取耦合的C2 -C3键的2-甲基乌头酸的旋转，和残基Lys 73是关键的PrpF功能。新发现的PrpF作为非PLP依赖性异构酶的功能，以及含有PrpD的细菌不需要PrpF的事实，表明作为顺乌头酸酶底物的2-甲基乌头酸异构体必须具有与合成的相同的立体化学结构PrpD。由此可见，AcnD产生的2-甲基乌头酸异构体不是乌头酸酶的底物，并且需要PrpF来产生正确的异构体。因此，PrpF的异构酶活性现在可以被视为2-甲基柠檬酸循环的一个组成部分。
In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 angstrom resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2 -C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle.