Cyanolysis and Azidolysis of Epoxides by Haloalcohol Dehalogenase: Theoretical Study of the Reaction Mechanism and Origins of Regioselectivity

Abstract

Haloalcohol dehalogenase HheC catalyzes the reversible dehalogenation of vicinal haloalcohols to form epoxides and free halides. In addition, HheC is able to catalyze the irreversible and highly regioselective ring-opening of epoxides with nonhalide nucleophiles, such as CN (-) and N 3 (-). For azidolysis of aromatic epoxides, the regioselectivity observed with HheC is opposite to the regioselectivity of the nonenzymatic epoxide-opening. This, together with a relatively broad substrate specificity, makes HheC a promising tool for biocatalytic applications. We have designed large quantum chemical models of the HheC active site and used density functional theory to study the reaction mechanism of the HheC-catalyzed ring-opening of ( R)-styrene oxide with the nucleophiles CN (-) and N 3 (-). Both the cyanolysis and the azidolysis reactions are shown to take place in a single concerted step. The results support the suggested role of the putative Ser132-Tyr145-Arg149 catalytic triad, where Tyr145 acts as a general acid, donating a proton to the substrate, and Arg149 interacts with Tyr145 and facilitates proton abstraction, while Ser132 positions the substrate and reduces the barrier for epoxide opening through interaction with the emerging oxyanion of the substrate. We have also studied the regioselectivity of ( R)-styrene oxide opening for both the cyanolysis and the azidolysis reactions. The employed active site model was shown to be able to reproduce the experimentally observed beta-regioselectivity of HheC. In silico mutations of various groups in the HheC active site model were performed to elucidate the important factors governing the regioselectivity.

DFT investigation on the reaction mechanism catalyzed by α-phosphomannomutase1 in protonated/deprotonated states

“…This approach was used to keep the various groups in place to ε=4 ε=8 ε=80 Barriers 7. 6 7.4 7.0 resemble the docked result as much as possible [26][27][28][29]. These fixed positions are indicated by arrows in the figures.…”

“…The dielectric constant chosen is ε=4, as is known that the standard value is used to model the protein surroundings. For all the models, the dielectric constant of ε=4 was used, the effect of using other dielectric constants are investigated in the first step in the protonated state, and results were shown to be quite insensitive to the choice of the constants (see Table 1) [26][27][28]. Hessians were calculated to confirm the nature of the stationary points, and were also used for evaluation of zero-point vibrational effects at the B3LYP/6-311G level.…”

Chemoenzymatic preparation of (S)-p-nitrostyrene oxide from p-nitrophenacyl bromide by recombinant Escherichia coli cells expressing a novel halohydrin dehalogenase

“…It showed higher activity under basic condition than under neutral or acid condition. It might be because the basic condition was beneficial to decrease the pK a of the catalytic residue Tyr and relay the abstracted proton [6], and thus facilitated proton abstraction from hydroxyl group of BNPE by re-SyHhdh to promote the forward reaction of the ring-closure of BNPE. The activity of re-SyHhdh kept increasing until 45°C, and decreased at higher temperature, especially at temperature above 55°C (Fig.…”

“…Tyr donates a proton to epoxide, and Arg as general acid interacts with Tyr. Subsequently, a nucleophile attacks the epoxide ring C β -atom producing the corresponding β-substituted alcohol [6,7].…”

“…HheC with excellent enzymatic properties has been studied and used extensively. Its crystal structures involved in the ring-closure and ring-opening reaction were available [6,14].…”