Baeyer–Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these ...atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (kcₐₜ = 1.9 s⁻¹, KM = 59 μM). The enzyme exhibits moderate enantioselectivity with α-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.
Easy virtue: Halohydrin dehalogenase is a highly promiscuous enzyme that can catalyze enantioselective epoxide ring opening with at least nine different anionic nucleophiles (see scheme). Its ...capacity to form carbon–nitrogen, carbon–oxygen, carbon–sulfur, and carbon–carbon bonds makes it possible to use this enzyme for the preparation of a range of highly enantioenriched β‐substituted alcohols or derivatives thereof, including cyanoalcohols, nitroalcohols, and oxazolidinones.
A novel one‐pot, two‐step, two‐enzyme cascade is described. Pro‐chiral α‐chloro ketones are stereoselectively reduced to the corresponding halohydrins as an intermediate by a biocatalytic hydrogen ...transfer process. The intermediate is transformed to the corresponding epoxide by a non‐enantioselective halohydrin dehalogenase. Thus, by combining a Prelog‐ or anti‐Prelog alcohol dehydrogenase with a non‐selective halohydrin dehalogenase, enantiopure (R)‐ as well as (S)‐epoxides were obtained.
In epoxide hydrolase from
Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this ...position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards
para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward
cis-disubstituted
meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted
cis-2,3-epoxybutane to (2
R,3
R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1
R,2
R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (
S)-epoxides and chiral vicinal diols in high yield and with excellent ee.
figure: see text The halohydrin dehalogenase from Agrobacterium radiobacter AD1 catalyzed the highly enantioselective and beta-regioselective azidolysis of (substituted) styrene oxides. By means of ...kinetic resolutions the remaining epoxide and the formed azido alcohol could be obtained in very high ee. In a large scale conversion, the decrease in yield and selectivity due to the uncatalyzed chemical side reaction could be overcome by slow addition of azide.
Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate. The recently solved X-ray structure of the enzyme shows that two ...tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step. The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis. Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations. Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation. The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations. The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants. This indicates that the effects of the mutations were cumulative. The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase. Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate. Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM.
Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 is a homotetrameric protein containing four tryptophan residues per subunit. The fluorescence properties of the enzyme are strongly ...influenced by halide binding. To examine the role of the tryptophans (W139, W192, W238, and W249) in halide binding and catalysis, they were individually mutated to a phenylalanine. All mutations, except for W238F, influenced the enzymatic properties. Mutating W192 to phenylalanine inactivated the enzyme and led to dissociation into dimers and monomers. In the structure of HheC, residue W139 and residue W249 from the opposite subunit are close to the active site of the enzyme. Substitution of W139 mainly affected K(m) values with all tested substrates and reduced the enantiopreference for p-nitro-2-bromo-1-phenylethanol. Replacing W249 increased both k(cat) and K(m) values with all tested substrates except for the (S)-enantiomer of p-nitro-2-bromo-1-phenylethanol, for which k(cat) was 3-fold decreased, resulting in a 6-fold increase of the enantioselectivity. Fluorescence measurements revealed that in the ligand-free state the intrinsic protein fluorescence of mutant W139F is higher than that of the wild-type enzyme, while the fluorescence intensity of mutants W238F and W249F was lower. The fluorescence intensities of the W238F and W249F enzymes were increased when they were unfolded or when bromide was added, whereas the fluorescence of mutant W139F was not increased by unfolding or addition of bromide. These results demonstrate that the fluorescence of residues W238 and W249 is partially quenched in the folded ligand-free state, and that W139 is completely quenched and acts as an energy acceptor for the other tryptophan residues as well. Changes of the maximum fluorescence emission wavelength of the HheC variants and the results of acrylamide quenching experiments confirmed that bromide binding induces a local conformational change around the active site, resulting in residue W139 and the quencher group being separated.
Halohydrin dehalogenases are bacterial enzymes that catalyse the reversible formation of epoxides from
vicinal halohydrins. A spectrophotometric assay for halohydrin dehalogenases based on the ...absorption difference between the halohydrin
para-nitro-2-bromo-1-phenylethanol and the epoxide
para-nitrostyrene oxide was developed. The enantioselectivity of ring-closure reactions catalysed by three different halohydrin dehalogenases could be estimated from the shape of progress curves. Evaluation of ring-opening reactions catalysed by halohydrin dehalogenase from
Agrobacterium radiobacter AD1 established that, in addition to Cl
− and Br
−, nucleophiles such as N
3
−, CN
− and NO
2
− are also accepted for the ring opening of
para-nitrostyrene oxide. The ring-opening reactions with these nucleophiles resulted in highly enantioselective kinetic resolutions, which expands the scope of synthetically valuable conversions catalysed by a halohydrin dehalogenase.
Graphic
PDF
