Search

Metallo-β-lactamase (MβL) is a eubacterial zinc metallo-hydrolase superfamily. Despite their well-known lactamase activities, MβL family members also have the ability to catalyze phosphotriester hydrolysis with different phosphotriesterase activities. In the present study, based on crystal structure comparisons of the related MβL members, a series of models was constructed and calculated using the density functional theory (DFT) method to explore the relationship between active-site changes and phosphotriesterase activities. These calculations show that the energetic barriers for phosphotriesterase activity are considerably reduced due to active-site differences, which describes an evolutionary trend for the development of phosphotriesterase activity in the MβL superfamily. The key event is the appearance of a specialized and negatively charged residue bridging both zinc ions, which plays the two important roles of maintaining charge balance and stabilizing the binuclear active-site structure. This pathway is also consistent with the evolutionary relationships determined by phylogenetic tree analysis using complete residue sequences. Our studies provide the first methodology to explore the development of a new enzyme activity within a superfamily, and to shed new light on understanding the catalytic mechanism from an evolutionary perspective., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a tungsten-dependent enzyme that catalyzes the oxidation of formaldehyde to formic acid. In the present study, quantum chemical calculations are used to elucidate the reaction mechanism of this enzyme. Several possible mechanistic scenarios are investigated with a large model of the active site designed on the basis of the X-ray crystal structure of the native enzyme. Based on the calculations, we propose a new mechanism in which the formaldehyde substrate binds directly to the tungsten ion. W(VI)=O then performs a nucleophilic attack on the formaldehyde carbon to form a tetrahedral intermediate. In the second step, which is calculated to be rate limiting, a proton is transferred to the second-shell Glu308 residue, coupled with a two-electron reduction of the tungsten ion. The calculated barriers for the mechanism are energetically very feasible and in relatively good agreement with experimental rate constants. Three other second-shell mechanisms, including one previously proposed based on experimental findings, are considered but ruled out because of their high barriers., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The reaction mechanism of the dinuclear zinc enzyme human renal dipeptidase is investigated using hybrid density functional theory. This enzyme catalyzes the hydrolysis of dipeptides and beta-lactam antibiotics. Two different protonation states in which the important active site residue Asp288 is either neutral or ionized were considered. In both cases, the bridging hydroxide is shown to be capable of performing the nucleophilic attack on the substrate carbonyl carbon from its bridging position, resulting in the formation of a tetrahedral intermediate. This step is followed by protonation of the dipeptide nitrogen, coupled with C-N bond cleavage. The calculations establish that both cases have quite feasible energy barriers. When the Asp288 is neutral, the hydrolytic reaction occurs with a large exothermicity. However, the reaction becomes very close to thermoneutral with an ionized Asp288. The two zinc ions are shown to play different roles in the reaction. Zn1 binds the amino group of the substrate, and Zn2 interacts with the carboxylate group of the substrate, helping in orienting it for the nucleophilic attack. In addition, Zn2 stabilizes the oxyanion of the tetrahedral intermediate, thereby facilitating the nucleophilic attack.