Structure and catalytic mechanism of LigI: insight into the amidohydrolase enzymes of cog3618 and lignin degradation.
Structure and catalytic mechanism of LigI: insight into the amidohydrolase enzymes of cog3618 and lignin degradation.
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DOI：
10.1021/bi300307b
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发表时间：
2012-04-24
期刊：
影响因子：
2.9
通讯作者：
Raushel FM
中科院分区：
文献类型：
--
作者：
Hobbs ME;Malashkevich V;Williams HJ;Xu C;Sauder JM;Burley SK;Almo SC;Raushel FM
少突鞘氨醇单胞菌的LIGI催化2-吡喃酮-4，6-二羧酸(PDC)在木质素降解过程中可逆地水解为4-草醛甲酸(OMA)和4-羧基-2-羟基-2-羟基木酸(CHM)。该蛋白是氨基水解酶超家族的成员。将该蛋白在大肠杆菌中表达，纯化至均一。纯化的重组酶不含结合的金属离子，向检测混合物中添加金属螯合剂或二价金属离子不影响产物的形成速度。这是酰胺水解酶超家族中第一种不需要二价金属离子就能发挥催化活性的酶。动力学常数分别为340 S−1和9.8×106M−1s−1，kcat/Km值。PH对动力学常数的依赖关系表明，对于PDC的水解，必须对单个活性中心残基进行去质子化。通过在18O标记的水中进行PDC的水解和随后的13C核磁共振分析来确定亲核攻击的位置。在反应产物存在的情况下，测定了野生型LIGI和D248A突变体的晶体结构，分辨率为1.9ó。PDC的C-8和C-11羧基分别与Arg-130和Arg-124通过离子对作用在活性中心内配位。水解水分子通过质子转移到Asp-248而被激活。通过与His-180、His-31和His-33的静电相互作用，内酯底物上的羰基被激活。
LigI from Sphingomonas paucimobilis catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate (OMA) and 4-carboxy-2-hydroxymuconate (CHM) in the degradation of lignin. This protein is a member of the amidohydrolase superfamily of enzymes. The protein was expressed in E. coli and then purified to homogeneity. The purified recombinant enzyme does not contain bound metal ions and the addition of metal chelators or divalent metal ions to the assay mixtures does not affect the rate of product formation. This is the first enzyme from the amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. The kinetic constants for the hydrolysis of PDC are 340 s−1 and 9.8 × 106 M−1s−1 for the values of kcat, and kcat/Km respectively. The pH dependence on the kinetic constants suggests that a single active site residue must be deprotonated for the hydrolysis of PDC. The site of nucleophilic attack was determined by conducting the hydrolysis of PDC in 18O-labeled water and subsequent 13C NMR analysis. The crystal structures of wild-type LigI and the D248A mutant in the presence of the reaction product were determined to a resolution of 1.9 Å. The C-8 and C-11 carboxylate groups of PDC are coordinated within the active site via ion pair interactions with Arg-130 and Arg-124, respectively. The hydrolytic water molecule is activated by a proton transfer to Asp-248. The carbonyl group of the lactone substrate is activated by electrostatic interactions with His-180, His-31 and His-33.
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影响因子：
2.9
作者：
Hall, Richard S.;Xiang, Dao Feng;Raushel, Frank M.
通讯作者：
Raushel, Frank M.
影响因子：
14.9
作者：
Tatusov, RL;Natale, DA;Koonin, EV
通讯作者：
Koonin, EV
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
DOI：
10.1093/bioinformatics/btq675
发表时间：
2011-02-01
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Smoot ME;Ono K;Ruscheinski J;Wang PL;Ideker T
通讯作者：
Ideker T
影响因子：
3
作者：
Pettersen, EF;Goddard, TD;Ferrin, TE
通讯作者：
Ferrin, TE