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.Halogenated aliphatics constitute an important class of environmental pollutants. Various microorganisms have evolved that are able to degrade some of these compounds and use them as sole sources of carbon and energy. Such organisms are of importance for bioremediation of polluted soil, groundwater, and wastewater. In most cases, specialized enzymes, designated dehalogenases, catalyze the cleavage of the carbonhalogen bonds, which is a key detoxification reaction. Hydrolytic dehalogenases have been studied extensively, which has resulted in detailed insight into the structure and mechanism of several enzymes of this class (8,33). For other dehalogenases, structural and mechanistic data are hardly available.Halohydrin dehalogenases, also referred to as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, occur in the degradation pathways of halopropanols and 1,2-dibromoethane, where they catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, yielding an epoxide, a proton, and a halide ion (7,22,30,31). These enzymes also efficiently catalyze the reverse reaction, the halogenation of epoxides, and the dehalogenation of vicinal chlorocarbonyls to hydroxycarbonyls (2, 14, 31). The interest in halohydrin dehalogenases increased when i...

Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic. These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen. Haloalcohol dehalogenases are related to the widespread family of NAD(P)Hdependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones. Our results reveal the ®rst structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site. Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors. Keywords: haloalcohol dehalogenase/SDR family/shortchain dehydrogenase/X-ray structure IntroductionDehalogenases are enzymes that are able to cleave carbonhalogen bonds . Structural characterization of haloalkane dehalogenases and haloacid dehalogenases demonstrated that these hydrolytic enzymes are evolutionarily related to widespread esterase and phosphatase families (Ollis et al., 1992;Hisano et al., 1996;Ridder and Dijkstra, 1999). Haloalcohol dehalogenases, also known as halohydrin dehalogenases or halohydrin hydrogen-halide lyases, cannot be classi®ed in these existing dehalogenase families. Instead, they show low sequence similarity to members of the short-chain dehydrogenase/reductase (SDR) family . This family contains redox enzymes that depend on NAD(P)H, which is bound in a characteristic dinucleotide binding fold (Rossmann fold) (Rossmann et al., 1974). They have a conserved catalytic triad of Ser, Tyr and Lys/Arg residues (Jo Èrnvall et al., 1995;Oppermann et al., 2003), which is also present in the haloalcohol dehalogenases . Many structures of shortchain dehydrogenases/reductases in complex with dinucleotides and substrates have revealed the structural details of the reactions catalyzed by them (Jo Èrnvall et al., 1995;Filling et al., 2002;Oppermann et al., 2003). In addition, the structure of a dinucleotide-binding transcription factor that lacked the catalytic tyrosine and thus oxidoreductase activity showed that the SDR fold also functions in nonenzymatic activities (Stammers et al., 2001).Haloalcohol dehalogenases catalyze the intramolecular displacement of a halogen by the vicinal hydroxyl group in 1,3-dichloro-2-propanol, yielding its ...

Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel small-intelligent strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.

[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.

Farmers’ organizations (FOs), such as associations, cooperatives, self-help and women’s groups, are common in developing countries and provide services that are widely viewed as contributing to income and productivity for small-scale producers. Here, we conducted a scoping review of the literature on FO services and their impacts on small-scale producers in sub-Saharan Africa and India. Most reviewed studies (57%) reported positive FO impacts on farmer income, but much fewer reported positive impacts on crop yield (19%) and production quality (20%). Environmental benefits, such as resilience-building and improved water quality and quantity were documented in 24% of the studies. Our analysis indicates that having access to markets through information, infrastructure, and logistical support at the centre of FO design could help integrate FOs into policy. Natural resource management should also be more widely incorporated in the services provided by FOs to mitigate risks associated with environmental degradation and climate change. Finally, farmers who are already marginalized because of poor education, land access, social status and market accessibility may require additional support systems to improve their capacities, skills and resources before they are able to benefit from FO membership.