Dynamic Kinetic Resolution Process Employing Haloalkane Dehalogenase

Westerbeek, Alja; Szymański, Wiktor; Feringa, Ben L.; Janssen, Dick B.

doi:10.1021/cs2003565

Cited by 22 publications

(18 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Furthermore, molecular dynamic simulations with LinB–substrate complexes showed that the preferred ( R )-enantiomer was more favorably bound in the active site. A dynamic kinetic resolution (DKR) procedure using polymer-based phosphonium bromide as the racemization catalyst led to highly enantioenriched α-hydroxyamides [94]. The reactions had to be conducted in a membrane reaction system to avoid inactivation of the enzyme by the racemizing polymer.…”

Section: Dehalogenasesmentioning

confidence: 99%

Inverting hydrolases and their use in enantioconvergent biotransformations

Schöber¹,

Faber²

2013

Trends in Biotechnology

View full text Add to dashboard Cite

show abstract

Section: Dehalogenasesmentioning

confidence: 99%

Inverting hydrolases and their use in enantioconvergent biotransformations

Schöber¹,

Faber²

2013

Trends in Biotechnology

View full text Add to dashboard Cite

show abstract

“…Haloalkane dehalogenases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/8/1/5.html) are hydrolytic enzymes that cleave carbon–halogen bonds in halogenated hydrocarbons. They have important applications in bioremediation , biosensing , biosynthesis , cellular imaging and protein analysis . These enzymes possess an active site deeply buried in a hydrophobic cavity between two domains.…”

Section: Introductionmentioning

confidence: 99%

Kinetics of binding of fluorescent ligands to enzymes with engineered access tunnels

et al. 2016

View full text Add to dashboard Cite

Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutations introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude, and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method's application to mediumthroughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkylenzyme intermediate during their catalytic cycle, such as a/b-hydrolases containing > 100 000 protein sequences based on the Pfam database.

show abstract

“…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.…”

mentioning

confidence: 99%

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

et al. 2017

View full text Add to dashboard Cite

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.

show abstract

Dynamic Kinetic Resolution Process Employing Haloalkane Dehalogenase

Cited by 22 publications

References 31 publications

Inverting hydrolases and their use in enantioconvergent biotransformations

Inverting hydrolases and their use in enantioconvergent biotransformations

Kinetics of binding of fluorescent ligands to enzymes with engineered access tunnels

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

Contact Info

Product

Resources

About