engleski

Expanding the scope of enantioselective halohydrin dehalogenases – group B

Halohydrin dehalogenases (HHDHs) possess an unnatural activity of introducing functionalities such as N3, CN, NO2 etc., into a molecule through the ring-opening reaction of epoxides. The enantioselectivity of HHDHs is substrate-dependent and not always high enough for synthetic applications. B-group of HHDHs has been neglected in the past, due to observed low enantioselectivity based on performance on a relatively limited number of substrates. Extensive screening of substrates on HheB2 from Mycobacterium sp. GP1 and HheB from Corynebacterium sp. N-1074 was performed. Several highly enantioselective reactions were discovered (E>200), with HheB showing higher enantioselectivity and activity toward larger panel of substrates compared to HheB2. Enzymes HheB and HheB2 are highly homologous ; they differ by only 4 residues. By using site-directed mutagenesis, residues 120 and 125 were found to be responsible for higher enantioselectivity of HheB compared to HheB2. Computational analysis supported experiments and provided evidence that kinetic and thermodynamic parameters of reactions within HheB enzymes are crucial in determining the observed enantioselectivities. Due to remarkable activity and enantioselectivity, B-group HHDHs emerged as a catalyst of choice for the synthesis of bulky tertiary alcohols, as shown in this work.

biocatalysis ; halohydrin dehalogenase ; epoxides ; kinetic resolution ; molecular modelling

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