Biocatalytic conversion of epoxides

Epoxides are attractive intermediates for producing chiral compounds. Important biocatalytic reactions involving epoxides include epoxide hydrolase mediated kinetic resolution, leading to the formation of diols and enantiopure remaining substrates, and enantioconvergent enzymatic hydrolysis, which gives high yields of a single enantiomer from racemic mixtures. Epoxides can also be converted by non-hydrolytic enantioselective ring opening, using alternative anionic nucleophiles; these reactions can be catalysed by haloalcohol dehalogenases. The differences in scope of these enzymatic conversions is related to their different catalytic mechanisms, which involve, respectively, covalent catalysis with an aspartate carboxylate as the nucleophile and non-covalent catalysis with a tyrosine that acts as a general acid-base. The emerging new possibilities for enantioselective biocatalytic conversion of epoxides suggests that their importance in green chemistry will grow.

COMETABOLIC DEGRADATION OF CHLOROALLYL ALCOHOLS IN BATCH AND CONTINUOUS CULTURES

The biodegradation of chloroallyl alcohols by pure and mixed bacterial cultures was investigated. Only 2-chloroallyl alcohol and cis- and trans-3-chloroallyl alcohol served as growth substrate for pure cultures. The other chloroallyl alcohols could be cometabolically degraded during growth on 2-chloroallyl alcohol. Cometabolic degradation of trichloroallyl alcohol, which was the most recalcitrant congener, by a Pseudomonas strain isolated on 2-chloroallyl alcohol resulted in 60% dechlorination. Efficient degradation of a mixture of chloroallyl alcohols in continuous culture could only be achieved in the presence of a satellite population. The mixed culture degraded 99% of the total chloroallyl alcohols added with 71% chloride release. The culture contained strains with a new catabolic potential. The results indicate the importance of mixed cultures and genetic adaptation for efficient chloroallyl alcohol removal

Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp strain m15-3

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp, strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better, Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp, strain m15-3 under the control of the heterologous promoters P-lac, P-dhlA, and P-trc. The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1 (pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.</p

Crystallographic and Fluorescence Studies of the Interaction of Haloalkane Dehalogenase with Halide Ions. Studies with Halide Compounds Reveal a Halide Binding Site in the Active Site

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 catalyzes the conversion of 1,2-dichloroethane to 2-chloroethanol and chloride without use of oxygen or cofactors. The active site is situated in an internal cavity, which is accesible from the solvent, even in the crystal. Crystal structures of the dehalogenase enzyme complexed with iodoacetamide, chloroacetamide, iodide, and chloride at pH 6.2 and 8.2 revealed a halide binding site between the ring NH's of two tryptophan residues, Trp-125 and Trp-175, located in the active site. The halide ion lies on the intersection of the planes of the rings of the tryptophans. The binding of iodide and chloride to haloalkane dehalogenase caused a strong decrease in protein fluorescence. The decrease could be fitted to a modified form of the Stern-Volmer equation, indicating the presence of fluorophors of different accessibilities. Halide binding was much stronger at pH 6.0 than at pH 8.2. Assuming ligand binding to Trp-125 and Trp-175 as the sole cause of fluorescence quenching, dissociation constants at pH 6.0 with chloride and iodide were calculated to be 0.49 +/- 0.04 and 0.074 +/- 0.007 mM, respectively. Detailed structural investigation showed that the halide binding site probably stabilizes the halide product as well as the negatively charged transition state occurring during the formation of the covalent intermediate

Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways

The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dha4, and part of invA. A 12-nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1,3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved dhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1,3-dichloropropene and 1,2-dibromoethane.</p

Identification of a Baeyer-Villiger monooxygenase sequence motif

Baeyer-Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C-C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXRXXXW(P/D). Studies with site-directed mutants of 4-hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD-dependent monooxygenases: the so-called flavin-containing monooxygenases (FMOs), the N-hydroxylating monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

Specificity and kinetics of haloalkane dehalogenase

Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols, The active site is buried inside the protein and lined with hydrophobic residues, The reaction proceeds via a covalent substrate-enzyme complex, This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase, The kinetic mechanism for the ''natural'' substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given, In general, brominated substrates had a lower K-m, but a similar k(cat) than the chlorinated analogs, The rate of C-Br bond cleavage was higher than the rate of C-CL bond cleavage, which is in agreement with the leaving group abilities of these halogens, The lower K-m for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower K-m for bromo-compounds, However, the rate-determining step in the conversion (k(cat)) of 1,2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different K-m but similar k(cat) values for these compounds, The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates

Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism

The sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacterium Rhodococcus sp. strain NCIMB13064 were compared. The 16S rRNA gene sequences showed less than 1% sequence divergence, and all haloalkane degraders clearly belonged to the genus Rhodococcus. All strains shared a completely conserved dhaA gene, suggesting that the dhaA genes were recently derived from a common ancestor. The genetic organization of the dhaA gene region in each of the haloalkane degraders was examined by hybridization analysis and DNA sequencing. Three different groups could be defined on the basis of the extent of the conserved dhaA segment. The minimal structure present in all strains consisted of a conserved region of 12.5 kb, which included the haloalkane-degradative gene cluster that was previously found in strain NCIMB13064. Plasmids of different sizes were found in all strains. Southern hybridization analysis with a dhaA gene probe suggested that all haloalkane degraders carry the dhaA gene region both on the chromosome and on a plasmid (70 to 100 kb). This suggests that an ancestral plasmid was transferred between these Rhodococcus strains and subsequently has undergone insertions or deletions. In addition, transposition events and/or plasmid integration may be responsible for positioning the dhaA gene region on the chromosome. The data suggest that the haloalkane dehalogenase gene regions of these gram-positive haloalkane-utilizing bacteria are composed of a single catabolic gene cluster that was recently distributed world-wide