Substrate Specificity of Fluoroacetate Dehalogenase: An Insight from Crystallographic Analysis, Fluorescence Spectroscopy, and Theoretical Computations

Abstract

The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 Å resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild‐type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate‐ and chloroacetate‐bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme–fluoroacetate complex. This is a stable but unreactive conformation, in which the small OCF angle is not suitable for the SN2 displacement of the F− ion. The cleavage of the CF bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the CO distance and the OCF angle are reasonable for the SN2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer CCl bond; this results in an increase of the activation energy despite the weaker CCl bond.

Theoretical prediction and experimental verification on enantioselectivity of haloacid dehalogenase l-DEX YL with chloropropionate

“…It is also observed in Figure 8 that the IFIE between Tyr60 and d-CPA is the most attractive of all the cases (WT/L, WT/D1, WT/D2, F60Y/D2, and F60Y/L), which is primarily due to the electrostatic interaction in light of the comparison between the MP2 and Hartree-Fock (HF) [30] results. Thus, we see that both Arg41 and Tyr60 can accept the chlorine atom in the complex of F60Y mutant and d-2-CPA, and expect that the activation energy would be reduced as in the cases of haloalkane dehalogenase [16] and fluoroacetate dehalogenase [18,19] owing is displayed by ball and stick. Carbon, chlorine, hydrogen, nitrogen and oxygen atoms are represented by cyan, green, white, blue, and red, respectively.…”

“…These experimental results indicate that Tyr12, Leu45 and Here, we focus on the mutation of Phe60 to Tyr60 (F60Y). Because the dehalogenation reaction is known [17,19] to depend strongly on the number of hydrogen bonds to a halide ion of substrate in the ester intermediate formation step, we constructed complex structure of the F60Y mutant with d-2-CPA (F60Y/D2) and optimized it with the ONIOM method. The reactant structure optimized is illustrated in Figure 7, in which Tyr60 is observed to make a hydrogen-bond-like interaction with the chlorine atom of CPA as Arg41 does.…”

“…A couple of theoretical studies on the mechanism of the specific substrate recognition in dehalogenases have been reported [19][20][21][22]. For example, the recognition mechanism of the fluoroacetate dehalogenase to substrates that have various halogen atoms (fluorine, chlorine and bromine) was accounted for by X-ray crystallographic analysis, fluorescence spectroscopy and QM calculations [19]. An enantioselectivity of haloalkane dehalogenase DbjA was analyzed by X-ray crystallographic analysis, thermodynamic analysis and MD simulations [20,21].…”

“…In addition to haloacid dehalogenase, the TS structures in haloalkane dehalogenase [16,17] and in fluoroacetate dehalogenase [18] have been computationally determined to elucidate their catalytic mechanisms. A couple of theoretical studies on the mechanism of the specific substrate recognition in dehalogenases have been reported [19][20][21][22]. For example, the recognition mechanism of the fluoroacetate dehalogenase to substrates that have various halogen atoms (fluorine, chlorine and bromine) was accounted for by X-ray crystallographic analysis, fluorescence spectroscopy and QM calculations [19].…”

Characterization of Wastewater Treatment Plant Microbial Communities and the Effects of Carbon Sources on Diversity in Laboratory Models

“…7, S5). The haloacetate dehalogenase (EC.3.8.1.3) from a Burkholderia species had been characterized [32] to catabolize chloroacetate to glycolate. Glycolate, in turn, can be oxidized to glyoxylate by glycolate oxidase and used as a carbon and energy source.…”