Enoyl-CoA hydratase (ECH),which is also known as crotonase,is the second requisite enzyme in theβ-oxidation pathway of fatty acid that catalyzes the syn hydration of α,β-unsaturated thiolester substrates.In this work,ECH-catalyzed hydration mechanisms of DAC-CoA and Crotonyl-CoA were investigated using density functional theory (DFT) methods.Geometrical structures were optimized using Gaussian 03 program at the B3LYP/6-31G(d,p) level of theory.Frequency calculations were performed with the 6-31G(d,p) basis set to obtain zero-point vibrational energies (ZPEs) and to confirm the nature of all the stationary points that have no imaginary frequency for the local minima and have only one imaginary frequency for the saddle points.The single-point calculations on the optimized geometries were further performed with 6-311 + +G(2d,2p) basis set to obtain more accurate energies.The polarizable-continuum model (PCM) with the dielectric constant of 4 was used to calculate the single point energies at 6-311 + +G(2d,p) level on all the optimized geometries to consider the effects of enzymatic environment that was not included in the computational model.Considering that B3LYP functional lacks the proper description of the long-range dispersion interactions,we further used the DFT-D3 program to calculate the empirical dispersion correction to correct the B3LYP energies.The final energies reported in this work are the single-point energies corrected for ZPEs,solvation and dispersion effects.The calculated results suggested that hydration proceeds through a stepwise mechanism,involving an enolate intermediate.Glu164 functions as the sole base/acid for catalysis.Although Glu144 is not directly involved in hydration,it induces the catalytic water molecule to locate an ideal orientation to attack the double bond of substrate by the hydrogen-bonding interaction.Crotonyl-CoA shows higher hydration activity than DAC-CoA.The backbone NH groups of Ala98 and Gly141 form an oxyanion hole with substrate carbonyl oxygen,which play key roles in binding substrate and stabilizing the generated transition states and intermediates.In addition,the hydrogen-bonding networks surrounding Glu144 and Glu164 are of great importance for active site arrangement.%采用DFT方法研究了烯酰基-辅酶A(ECH)催化的4-(N,N-二甲氨基)-肉桂酰-辅酶A(DAC-CoA)和巴豆酰基-辅酶A (Crotonyl-CoA)水合反应.计算表明:水合反应以分步机理进行,经历一个烯醇负离子中间体.Glu164残基作为唯一的催化碱/酸参与水合反应,而Glu144虽然没有直接参与反应,但是它能通过氢键作用诱使水分子以合适的朝向活化底物.Crotonyl-CoA底物的水加成活性高于DAC-CoA.Ala98和Glyl41与底物羰基之间的氢键作用既有利于底物的准确结合,也能有效稳定反应中形成的过渡态和中间体.另外,Glu144和Glu164周围的氢键网络对于合理维持活性位点排布进而有效促进底物活化也很重要.
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