Haloalkane dehalogenases from five sources were heterologously expressed in Escherichia coli, isolated, and tested for their ability to achieve kinetic resolution of racemic a-bromoamides, which are important intermediates used in the preparation of bioactive compounds. To explore the substrate scope, fourteen a-bromoamides, with different Caand N-substituents, were synthesized. Catalytic activity towards eight substrates was found, and for five of these compounds the conversion proceeded with a high enantioselectivity (E value > 200). In all cases, the (R)-a-bromoamide is the preferred substrate. Conversions on a preparative scale with a catalytic amount of enzyme (enzyme:substrate ratio less 1:50 w/w) were all completed within 17-46 h and optically pure a-bromoamides and a-hydroxyamides were isolated with good yields (31-50%). Substrate docking followed by molecular dynamics simulations indicated that the high enantioselectivity results from differences in the percentage of the time in which the substrate enantiomers are bound favourably for catalysis. For the preferred (R)-substrates, the angle between the attacking aspartate oxygen atom of the enzyme, the attacked carbon atom of the substrate, and the displaced halogen atom, is more often in the optimal range (> 1578) for reactivity. This can explain the observed enantioselectivity of LinB dehalogenase in a kinetic resolution experiment.
Lacticin 3147 is a lantibiotic with seven lanthionine bridges across its two component peptides, Ltnα and Ltnβ. Although it has been proposed that the eponymous lanthionine and (β-methyl)lanthionine (Lan and meLan) bridges present in lantibiotics make an important contribution to protecting the peptides from thermal or proteolytic degradation, few studies have investigated this link. We have generated a bank of bioengineered derivatives of lacticin 3147, in which selected bridges were removed or converted between Lan and meLan, which were exposed to high temperature or proteolytic enzymes. Although switching Lan and meLan bridges has variable consequences, it was consistently observed that an intact N-terminal lanthionine bridge (Ring A) confers Ltnα with enhanced resistance to thermal and proteolytic degradation.
The first dynamic kinetic resolution process with a haloalkane dehalogenase is described, allowing the efficient preparation of enantiopure α-hydroxyamides from racemic α-bromoamides. A simple membrane reactor is used to separate the enzyme from the nonsoluble, polymer-based, and metal-free racemizing agent. A model substrate, N-phenyl-2-bromopropionamide, was converted to (S)-N-phenyl-2-hydroxypropionamide either with 63% yield and 95% e.e or with 78% yield and 88% e.e.
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