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Theoretical Studies on the Binding Mode and Reaction Mechanism of TLP Hydrolase kpHIUH.

ABSTRACT: In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based on this binding conformation, density functional theory calculations indicated that the optimal catalytic mechanism consists of two stages: (1) the lytic water molecule is deprotonated by His92 and carries out nucleophilic attack on C6=O of 5-HIU, resulting in an oxyanion intermediate; (2) by accepting a proton transferred from His92, C6-N5 bond is cleaved to completes the catalytic cycle. The roles of His7, His92, Ser108 and Arg49 in the catalytic reaction were revealed and discussed in detail.

SUBMITTER: Wang X

PROVIDER: S-EPMC8272043 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Theoretical Studies on the Binding Mode and Reaction Mechanism of TLP Hydrolase kpHIUH.

Wang Xixi X Shan Jiankai J Liu Wei W Li Jing J Tan Hongwei H Li Xichen X Chen Guangju G

Molecules (Basel, Switzerland) 20210625 13

In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based ...[more]

PMID: 34202153

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Project description:Retaining glycoside hydrolases use acid/base catalysis with an enzymatic acid/base protonating the glycosidic bond oxygen to facilitate leaving-group departure alongside attack by a catalytic nucleophile to form a covalent intermediate. Generally, this acid/base protonates the oxygen laterally with respect to the sugar ring, which places the catalytic acid/base and nucleophile carboxylates within about 4.5-6.5 Å of each other. However, in glycoside hydrolase (GH) family 116, including disease-related human acid β-glucosidase 2 (GBA2), the distance between the catalytic acid/base and the nucleophile is around 8 Å (PDB: 5BVU) and the catalytic acid/base appears to be above the plane of the pyranose ring, rather than being lateral to that plane, which could have catalytic consequences. However, no structure of an enzyme-substrate complex is available for this GH family. Here, we report the structures of Thermoanaerobacterium xylanolyticum β-glucosidase (TxGH116) D593N acid/base mutant in complexes with cellobiose and laminaribiose and its catalytic mechanism. We confirm that the amide hydrogen bonding to the glycosidic oxygen is in a perpendicular rather than lateral orientation. Quantum mechanics/molecular mechanics (QM/MM) simulations of the glycosylation half-reaction in wild-type TxGH116 indicate that the substrate binds with the nonreducing glucose residue in an unusual relaxed 4C1 chair at the -1 subsite. Nevertheless, the reaction can still proceed through a 4H3 half-chair transition state, as in classical retaining β-glucosidases, as the catalytic acid D593 protonates the perpendicular electron pair. The glucose C6OH is locked in a gauche, trans orientation with respect to the C5-O5 and C4-C5 bonds to facilitate perpendicular protonation. These data imply a unique protonation trajectory in Clan-O glycoside hydrolases, which has strong implications for the design of inhibitors specific to either lateral protonators, such as human GBA1, or perpendicular protonators, such as human GBA2.

Project description:BACKGROUND: Protein-DNA interactions are essential for fundamental biological activities including DNA transcription, replication, packaging, repair and rearrangement. Proteins interacting with DNA can be classified into two categories of binding mechanisms - sequence-specific and non-specific binding. Protein-DNA specific binding provides a mechanism to recognize correct nucleotide base pairs for sequence-specific identification. Protein-DNA non-specific binding shows sequence independent interaction for accelerated targeting by interacting with DNA backbone. Both sequence-specific and non-specific binding residues contribute to their roles for interaction. RESULTS: The proposed framework has two stage predictors: DNA-binding residues prediction and binding mode prediction. In the first stage - DNA-binding residues prediction, the predictor for DNA specific binding residues achieves 96.45% accuracy with 50.14% sensitivity, 99.31% specificity, 81.70% precision, and 62.15% F-measure. The predictor for DNA non-specific binding residues achieves 89.14% accuracy with 53.06% sensitivity, 95.25% specificity, 65.47% precision, and 58.62% F-measure. While combining prediction results of sequence-specific and non-specific binding residues with OR operation, the predictor achieves 89.26% accuracy with 56.86% sensitivity, 95.63% specificity, 71.92% precision, and 63.51% F-measure. In the second stage, protein-DNA binding mode prediction achieves 75.83% accuracy while using support vector machine with multi-class prediction. CONCLUSION: This article presents the design of a sequence based predictor aiming to identify sequence-specific and non-specific binding residues in a transcription factor with DNA binding-mechanism concerned. The protein-DNA binding mode prediction was introduced to help improve DNA-binding residues prediction. In addition, the results of this study will help with the design of binding-mechanism concerned predictors for other families of proteins interacting with DNA.

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Theoretical Studies on the Binding Mode and Reaction Mechanism of TLP Hydrolase kpHIUH.

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Theoretical Studies on the Binding Mode and Reaction Mechanism of TLP Hydrolase <i>kp</i>HIUH.

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