Crystal Structure of Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 Å Resolution: Dynamics of Catalytic Residues
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- 作者：Aaron J. Oakley ; Martin Klva
//pubs.acs.org/images/entities/ncaron.gif" border="0">a ; Michal Otyepka ; Yuji Nagata ; Matthew C. J. Wilce ; and Ji
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：February 3, 2004
- 年：2004
- 卷：43
- 期：4
- 页码：870 - 878
- 全文大小：649K
- 年卷期：v.43,no.4(February 3, 2004)
- ISSN：1520-4995
文摘
We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonaspaucimobilis UT26, at 0.95 Å resolution. The data have allowed us to directly observe the anisotropicmotions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displaysa high degree of disorder. It has been modeled in two conformations, one similar to that observed previously(conformation A) and one strained (conformation B) that approached the catalytic base (His272). Thestrain in conformation B was mainly in the C
-C
-C
angle (126
) that deviated by 13.4 from the"ideal" bond angle of 112.6. On the basis of these observations, we propose a role for the charge stateof the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized thatdouble-protonation of the catalytic base (His272) reduces the distance between the side-chain of thisresidue and that of the Asp108. The results of molecular dynamics simulations were consistent with thestructural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce thedistance between the side-chains of the residues in question, although the simulations failed to demonstratethe same degree of strain in the Asp108 C-C-C angle. Instead, the changes in the molecular dynamicsstructures were distributed over several bond and dihedral angles. Quantum mechanics calculations onLinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active siteconformations and protonation states were most likely to result in catalysis. It was shown that His272singly protonated at N
1 and Asp108 in conformation A gave the most exothermic reaction (
H = -22kcal/mol). With His272 doubly protonated at N1 and N
2, the reactions were only slightly exothermic orwere endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C-C-Cangle changed during the reaction and the Asp108 moved to conformation A. The results presented hereindicate that the positions of the catalytic residues and charge state of the catalytic base are important fordetermining reaction energetics in LinB.
-C-C angle (126) that deviated by 13.4 from the"ideal" bond angle of 112.6. On the basis of these observations, we propose a role for the charge stateof the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized thatdouble-protonation of the catalytic base (His272) reduces the distance between the side-chain of thisresidue and that of the Asp108. The results of molecular dynamics simulations were consistent with thestructural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce thedistance between the side-chains of the residues in question, although the simulations failed to demonstratethe same degree of strain in the Asp108 C-C-C angle. Instead, the changes in the molecular dynamicsstructures were distributed over several bond and dihedral angles. Quantum mechanics calculations onLinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active siteconformations and protonation states were most likely to result in catalysis. It was shown that His272singly protonated at N1 and Asp108 in conformation A gave the most exothermic reaction (H = -22kcal/mol). With His272 doubly protonated at N1 and N2, the reactions were only slightly exothermic orwere endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C-C-Cangle changed during the reaction and the Asp108 moved to conformation A. The results presented hereindicate that the positions of the catalytic residues and charge state of the catalytic base are important fordetermining reaction energetics in LinB.