Naslov
Biocatalytic properties of group B halohydrin dehalogenases in nucleophilic ring-opening reactions of epoxides

Autori
Mehić, Emina ; Findrik Blažević, Zvjezdana ; Majerić Elenkov, Maja

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
GRC Biocatalysis, Making Biocatalysis More Predictive: Accelerating Biocatalytic Solutions to Synthetic Challenges

Mjesto i datum
Manchester (NH), Sjedinjene Američke Države, 10.07.2022. - 15.07.2022

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
Biocatalyst, HHDH, enantioselectivity

Sažetak
Halohydrin dehalogenases (HHDHs) belong to a distinct group of enzymes catalyzing the removal of a halide ion and a proton from a vicinal halohydrin with the formation of an epoxide. Moreover, these enzymes can catalyze the epoxide ring-opening reactions with a range of anionic nucleophiles affording β-substituted alcohols as products. Based on sequence similarities, they are divided into 7 phylogenetic groups: A, B, C, D, E, F and G. Activity, enantioselectivity and enantiopreference are dependent on the type of enzyme and the substrate structure. HheC from Agrobacterium radiobacter is the most studied because of its high enantioselectivity. In order to expand the catalytic relevance of HHDHs we focus our attention to an enzyme from B group on which a small amount of research has been done. There are two similar enzymes in group B, one found in Corynebacterium sp. N1074 (HheB) and the other in Mycobacterium sp. GP1 (HheB2). They share high sequence identity (95%) with only 4 amino acid substitutions HheB/HheB2: F36/I36, T120/A120, C124/Y124, H125/Q125. HheB exhibits higher enantioselectivity than does HheB2. HheB2 has been previously characterized as a non-enantioselective enzyme, and neglected as a biocatalyst. In this work, HheB2 and HheB were investigated in the ring-opening reaction on a set of 21 structurally different aliphatic and aromatic epoxides using sodium azide as a nucleophile. To gain more insight into the difference of their enantioselectivity, mutants HheB2-F36I, HheB2- T120A, HheB2-C124Y, HheB2-H125Q were constructed and also tested. We examined nucleophiles that are accepted in the active site of B-type and used kinetics measurements to determine enzyme affinity and inhibition concentration for all accepted nucleophiles. The screening confirmed low to moderate enantioselectivity of HheB2 towards monosubstituted epoxides, however high activity and enantioselectivity in conversion of 2, 2- disubstituted (E-values up to >200). Enantioselectivity of HheB was slightly higher than HheB2 in the majority of cases. T120A mutation has been shown to be a key for increasing enantioselectivity in aliphatic epoxides. Among all tested substrates, disubstituted benzyl epoxides showed great potential for producing enantiomerically pure β-substituted alcohols. We showed that bicyclic structures as well, can be accepted into the active site of B-type with high E values (>200). Screening of nucleophiles confirmed that azide, cyanide, nitrite, cyanate and thiocyanate are accepted into the active site of the enzyme. A kinetics measurement showed that reaction rates are highest with azide ion and it is the only nucleophile that does not cause enzyme inhibition even at concentrations of 100 mM. Four other nucleophiles (CN-, NO2-, OCN- and SCN-) cause inhibition at concentrations between 5 and 25 mM.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Kemijsko inženjerstvo

POVEZANOST RADA