Enantioselectivity of Haloalkane Dehalogenases and its Modulation by Surface Loop Engineering

Abstract

In the loop: Engineering of the surface loop in haloalkane dehalogenases affects their enantiodiscrimination behavior. The temperature dependence of the enantioselectivity (lnE versus 1/T) of β‐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 α‐bromoesters.

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

“…[25] The HLD familyb elongst ot he superfamilyo fa/b-hydrolases and can be further subdivided into three subfamilies:H LD-I, HLD-II, and HLD-III. [26] HLDs are versatile biocatalysts with potentiala pplications in bioremediation, [27,28] biocatalysis, [29,30] biosensing, [31][32][33] and cell imaging. [34][35][36] During the past three decades, 23 HLDs have been identified and (at least partially) biochemically characterized.…”

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

“…Haloalkane dehalogenases (HLDs;E C3.8.1.5) catalyze the hydrolytic cleavage of carbon-halogen bonds by an S N 2 mechanism (see Figure 1inthe Supporting Information). [11,12] They are useful for the enantiodiscrimination of b-bromoalkanes, a-bromoesters,and a-bromoamides, [13][14][15][16] thus yielding av ariety of enantiomerically pure intermediates that are useful for the synthesis of drugs and other bioactive compounds. [14,17,18] HLDs are good model systems for studying the principles of enantioselectivity because individual family members show different enantioselectivity and their 3D structure has been determined at atomic resolution.…”

“…Thee ntropic component (which is around 83 %a s large as the enthalpic component) also contributes significantly to the enantioselectivity of DbjA. [13] Thelarge entropic component is attributed to the high hydration of the binding site: [21] Water promotes hydrophobic interactions between awall in the active site and the alkyl chains of the enantiomers in such aw ay that (R)-2-BP binds exclusively in ar eactive mode but (S)-2-BP adopts both reactive and nonreactive binding modes. [13] Thee ntropic contribution for weakly enantioselective,l ess hydrated DhaA is much smaller.…”

Evaluation of Pseudoenantiomeric Mixed Carbonates as Efficient Fluorogenic Probes for Enantioselectivity Screening

“…[28] Only af ew reportsh ave described the effective kinetic resolution of butan-2-ol. [29] Determining the enantiomeric excesso f butan-2-ol was also problematic.W ith our method, derivatization of butan-2-ol with 4-methylumbelliferone resulted in very good chiralH PLC separation. The retention times between enantiomers differed by 78 s. The use of fluorophores as derivatizing agentsi sc ommon to achieve effective separation of compounds on chromatographic columns.…”