Jeffrey H. Lutje Spelberg

Discovery of a thermostable Baeyer-Villiger monooxygenase by genome mining.

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.

Halohydrin Dehalogenases Are Structurally and Mechanistically Related to Short-Chain Dehydrogenases/Reductases

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.

Catalytic Promiscuity of Halohydrin Dehalogenase and its Application in Enantioselective Epoxide Ring Opening

The catalytic promiscuity of some enzymes is thought to play an important role in the evolution of new catalytic activities, and can be used to apply enzymes in unnatural reactions of synthetic importance. However, unnatural enzyme-catalyzed reactions are often slow and most documented cases concern hydrolytic conversions instead of synthetic reactions. The enantioselectivity of promiscuous enzymes also may be low. Here, we report that an unusual dehalogenase that has recently been explored structurally and mechanistically can accept at least nine different anionic nucleophiles in enantioselective ACHTUNGTRENNUNGepoxide ring-opening reactions that are catalyzed by this enzyme. This enables the preparation of a broad range of highly enantioenriched b-substituted alcohols and epoxides by kinetic resolution. Halohydrin dehalogenase (HheC) from the epichlorohydrin degrading bacterium Agrobacterium radiobacter AD1 catalyzes the dehalogenation of 1,3-dichloropropanol and 1-chloropropane-2,3-diol to produce an epoxide and HCl. Recently solved X-ray structures of HheC with various ligands revealed that the active site consists of a binding site for the epoxide and a ACHTUNGTRENNUNGspacious halide-binding pocket, and provided insight into the catalytic mechanism and cause of enantioselectivity of the enzyme. The dehalogenase also catalyzes the reverse reACHTUNGTRENNUNGaction: ring opening of epoxides. Nucleophilic epoxide ring opening is a powerful way to produce b-functionalized alcohols, which can be applied in the synthesis of pharmaceuticals and biologically active compounds. Considering that such compounds are often required as pure enantiomers, enantioand regioselective epoxide ringopening reactions have been studied intensively, for example with the use of chiral metal catalysts. So far, few nucleophiles have been used for epoxide ring opening, apart from water, halides, azide, and cyanide. We explored the catalytic versatility of HheC in epoxide ring opening by determining the products that are formed upon incubation of the enzyme with a series of different nucleophiles and 1,2-epoxybutane (5 mm) in buffer. Conversion was observed with Br , Cl , I , CN , NO2 , N3 , OCN , SCN , and HCOO . No reaction (epoxide conversion less than 0.05 mmolmin mg ) occurred with nonanionic nucleophiles (primary alcohols and amines), and with the sulfur-containing compounds H2S, SO3 2 , SO4 2 , and S2O5 2 . Acetic acid, chloro-, bromo-, and iodoacetic acid, 2-chloropropionic acid, malonic acid, PO4 2 , BO3 3 , PO4 2 , CO3 2 , H2O2, ClO4 , and F were also not accepted. A low conversion rate (0.1 mmolmin mg ) was obtained with NO3 . This indicates that the enzyme can accept a range of monovalent anions with a linear shape. In order to determine the rate of 1,2-epoxybutane conversion and monitor product formation, epoxybutane was incubated in a buffered solution at pH 7.5 with each accepted nu-

Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

Abstract The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in reasonable yield. The enantioselectivity (E-value) of the resolution was calculated from progress curves for styrene oxide (E=16.2) and para-chlorostyrene oxide (E=32.2).

Exploration of the biocatalytic potential of a halohydrin dehalogenase using chromogenic substrates

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 NO2- 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

A tandem enzyme reaction to produce optically active halohydrins, epoxides and diols

Abstract The recombinant halohydrin dehalogenase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure halohydrins and epoxides by kinetic resolution. By adding an excess of the recombinant epoxide hydrolase from the same organism the reversible conversion was drawn to completion. Halohydrins such as (S)-2,3-dichloro-1-propanol (E>100) and (S)-2-chloro-1-phenylethanol (E=73) were obtained with an enantiomeric excess of higher than 99%. This is a novel biocatalytic route for obtaining enantiomerically pure aromatic halohydrins and epoxides.

Characterization of the Gene Cluster Involved in Isoprene Metabolism in Rhodococcus sp. Strain AD45

The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in Rhodococcus sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation: a glutathione S-transferase with activity towards 1,2-epoxy-2-methyl-3-butene (isoI) and a 1-hydroxy-2-glutathionyl-2-methyl-3-butene dehydrogenase (isoH). Furthermore, a gene encoding a second glutathione S-transferase was identified (isoJ). The isoJ gene was overexpressed in Escherichia coli and was found to have activity with 1-chloro-2,4-dinitrobenzene and 3,4-dichloro-1-nitrobenzene but not with 1, 2-epoxy-2-methyl-3-butene. Downstream of isoJ, six genes (isoABCDEF) were found; these genes encoded a putative alkene monooxygenase that showed high similarity to components of the alkene monooxygenase from Xanthobacter sp. strain Py2 and other multicomponent monooxygenases. The deduced amino acid sequence encoded by an additional gene (isoG) showed significant similarity with that of alpha-methylacyl-coenzyme A racemase. The results are in agreement with a catabolic route for isoprene involving epoxidation by a monooxygenase, conjugation to glutathione, and oxidation of the hydroxyl group to a carboxylate. Metabolism may proceed by fatty acid oxidation after removal of glutathione by a still-unknown mechanism.

Improved stability of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by replacement of cysteine residues

Halohydrin dehalogenase from Agrobacterium radiobacterAD1 is a homo-tetrameric protein containing three cysteines per 28 kDa subunit. Under oxidizing conditions the enzyme was found to be susceptible to inactivation which could be prevented by the addition of -mercaptoethanol or glycerol. Gel filtration experiments and SDS-PAGE analysis revealed that inactivation coincided with monomerization and intramolecular disulfide bond formation. To identify the cysteine residues involved in the inactivation process, a set of cysteine mutant enzymes was constructed. All the purified mutants (C30A, C153S, C229A and C153S/C229A) showed a similar activity as wild-type enzyme, indicating that no cysteine is directly involved in catalysis. The C153S and C30A mutants displayed a higher stability than wild-type enzyme, whereas mutating Cys229 resulted in decreased enzyme stability. SDS-PAGE analysis showed that in wild-type enzyme Cys30-Cys229 and Cys153-Cys229 disulfide bonds were readily formed while almost no formation of the Cys30-Cys153 disulfide bond could be observed. From this, it was concluded that all three cysteine residues are involved in the enzyme inactivation process. The importance of the improved stability of the C153S and C30A mutant enzymes was demonstrated by performing kinetic resolution experiments with racemic 2-chloro-1-phenylethanol, which resulted in higher enantiomeric excess values of the remaining halohydrin when compared to conversions catalyzed by wild-type enzyme.

Biocatalytic Potential of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 and a Mutant with Enhanced Enantioselectivity

Optically pure epoxides are useful synthons for a variety of biologically active compounds. The epoxide hydrolase obtained from Agrobacterium radiobacter AD1 hydrolyses racemic aryl epoxides with moderate and aliphatic epoxides with low enantioselectivity. The three-dimensional structure of this enzyme indicates that two tyrosine residues interact with the epoxide oxygen. Mutating one of these, tyrosine 215, to a phenylalanine (Y215F) resulted in an enzyme with increased enantioselectivity towards aryl epoxides. The relatively strong decrease in activity towards the remaining enantiomers makes this enzyme a much better biocatalyst than the wild-type enzyme for the preparation of optically pure (S)-styrene oxide derivatives.

The enantioselectivity of haloalkane dehalogenases

Two haloalkane dehalogenases were tested for their ability to perform kinetic resolutions of a series of racemic substrates and to convert meso substrates enantioselectively. For the kinetic resolutions E-values of up to 9 were measured and, in the conversions of the meso substrates, products were obtained with an enantiomeric excess of up to 47%. A kinetic analysis revealed that despite modest overall chiral recognition (expressed as E-values), there are large differences between the K-m values (>100 fold) of two enantiomeric substrates but that these differences are compensated by correspondingly large differences in k(cat)