Veronika Stepankova

Enantioselectivity of Haloalkane Dehalogenases and its Modulation by Surface Loop Engineering

Engineering of the surface loop in haloalkane dehalogenases affects their enantiodiscrimination behavior. The temperature dependence of the enantioselectivity (lnE versus 1/T) of -bromoalkanes by haloalkane dehalogenases is reversed (red data points) by deletion of the surface loop; the selectivity switches back when an additional single-point mutation is made. This behavior is not observed for -bromoesters.

Dynamics and hydration explain failed functional transformation in dehalogenase design

We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.

Organic co-solvents affect activity, stability and enantioselectivity of haloalkane dehalogenases.

Haloalkane dehalogenases are microbial enzymes with a wide range of biotechnological applications, including biocatalysis. The use of organic co‐solvents to solubilize their hydrophobic substrates is often necessary. In order to choose the most compatible co‐solvent, the effects of 14 co‐solvents on activity, stability and enantioselectivity of three model enzymes, DbjA, DhaA, and LinB, were evaluated. All co‐solvents caused at high concentration loss of activity and conformational changes. The highest inactivation was induced by tetrahydrofuran, while more hydrophilic co‐solvents, such as ethylene glycol and dimethyl sulfoxide, were better tolerated. The effects of co‐solvents at low concentration were different for each enzyme‐solvent pair. An increase in DbjA activity was induced by the majority of organic co‐solvents tested, while activities of DhaA and LinB decreased at comparable concentrations of the same co‐solvent. Moreover, a high increase of DbjA enantioselectivity was observed. Ethylene glycol and 1,4‐dioxane were shown to have the most positive impact on the enantioselectivity. The favorable influence of these co‐solvents on both activity and enantioselectivity makes DbjA suitable for biocatalytic applications. This study represents the first investigation of the effects of organic co‐solvents on the biocatalytic performance of haloalkane dehalogenases and will pave the way for their broader use in industrial processes.

DspA from Strongylocentrotus purpuratus: The first biochemically characterized haloalkane dehalogenase of non-microbial origin

Haloalkane dehalogenases are known as bacterial enzymes cleaving a carbon-halogen bond in halogenated compounds. Here we report the first biochemically characterized non-microbial haloalkane dehalogenase DspA from Strongylocentrotus purpuratus. The enzyme shows a preference for terminally brominated hydrocarbons and enantioselectivity towards β-brominated alkanes. Moreover, we identified other putative haloalkane dehalogenases of eukaryotic origin, representing targets for future experiments to discover dehalogenases with novel catalytic properties.

Comparison of catalysis by haloalkane dehalogenases in aqueous solutions of deep eutectic and organic solvents

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds in diverse halogenated hydrocarbons and are attractive catalysts for sustainable biotechnologies. However, their use in industrial processes is limited due to the poor water solubility of their substrates and the tendency of the substrates to undergo abiotic hydrolysis. Here we systematically and critically compare the performance of three haloalkane dehalogenases, DbjA, DhaA and LinB, in aqueous solutions of the deep eutectic solvent ethaline, its components (ethylene glycol and choline chloride), and two organic solvents (methanol and acetone). Each of the solvents had different effects on the activity of each enzyme. Haloalkane dehalogenase DhaA was found to be the most tolerant to ethaline, retaining 21% of its reference activity even in solutions containing 90% (v/v) of ethaline. However, dissolution in 75% (v/v) ethylene glycol, 50% (v/v) methanol, or 25% (v/v) acetone caused almost total loss of DhaA activity. In contrast, the activities of DbjA and LinB were higher in ethylene glycol than in ethaline, and moreover the activity of DbjA was 1.5 times higher in 50% (v/v) ethylene glycol than in pure buffer. Interestingly, the enantioselectivity of 2-bromopentane hydrolysis catalysed by DbjA increased more than 4-fold in the presence of ethaline or ethylene glycol. Our results demonstrate that ethylene glycol and an ethylene glycol-based deep eutectic solvent can have beneficial effects on catalysis by haloalkane dehalogenases, broadening their usability in “green” biotechnologies.

Different Structural Origins of the Enantioselectivity of Haloalkane Dehalogenases toward Linear β‐Haloalkanes: Open–Solvated versus Occluded–Desolvated Active Sites

The enzymatic enantiodiscrimination of linear β-haloalkanes is difficult because the simple structures of the substrates prevent directional interactions. Herein we describe two distinct molecular mechanisms for the enantiodiscrimination of the β-haloalkane 2-bromopentane by haloalkane dehalogenases. Highly enantioselective DbjA has an open, solvent-accessible active site, whereas the engineered enzyme DhaA31 has an occluded and less solvated cavity but shows similar enantioselectivity. The enantioselectivity of DhaA31 arises from steric hindrance imposed by two specific substitutions rather than hydration as in DbjA.