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A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhLA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhLA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.Xanthobacter spp. are nitrogen-fixing bacteria that are able to grow autotrophically with a mixture of hydrogen and oxygen as an energy source (33). A member of this genus that is able to utilize several halogenated hydrocarbons as carbon sources has been isolated (15). The organism was obtained from an enrichment culture with 1,2-dichloroethane, which is an environmentally important compound with a production volume larger than that of any other industrial halogenated chemical. The 1,2-dichloroethane-degrading bacterium, designated strain GJ10, was found to degrade 1,2-dichloroethane via 2-chloroethanol, 2-chloroacetaldehyde, and chloroacetic acid to glycolate ( Fig. 1) (13, 14). The dehalogenation steps in this sequence were found to be catalyzed by two different hydrolytic dehalogenases (14,17).Conversion of 1,2-dichloroethane was mediated by a haloalkane dehalogenase. This was the first enzyme found to catalyze hydrolytic dehalogenation of chlorinated hydrocarbons. The protein has been purified (17) and crystallized (26), and its three-dimensional structure is now under study. Chloroacetic acid hydrolysis was found to be mediated by a different enzyme. This haloacid dehalogenase has not been purified from strain GJ10, but much information is available about other dehalogenases of this class (22).So far, haloalkane dehalogenases are the only enzymes known to be capable of direct hydrolytic dehalogenation of chlorinated and brominated hydrocarbons, without the requirement for coenzymes or oxygen. The enzyme of X. autotrophicus GJ10 is constitutively expressed to 2 to 3% of the soluble cellular protein (13, 17). It has a remarkably broad substrate range which includes terminally halogenated alkanes with chain lengths up to 4 carbons for chlorinated and up to at least 10 carbons for brominated alkanes. Other haloalkane dehalogenases of broad substrate range have been found in gram-positive haloalkane-utilizing bacteria (11,28,35 So far, no information is available about the genetics of haloalkane-utilizing organisms. Since the system is attractive both for st...

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ1O was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2S04 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The Hydrolytic dehalogenases are key enzymes in the detoxification of aliphatic halogenated hydrocarbons. They catalyze the cleavage of carbon-halogen bonds through a nucleophilic substitution by water to yield an alcohol. At least two distinct groups can be recognized with respect to their substrate ranges: haloalkane dehalogenases hydrolyze halogenated alkanes, whereas haloacid dehalogenases are active with short-chain 2-halogenated carboxylic acids.Of the 2-haloacid dehalogenases (E.C. 3.8.1.2), a number of enzymes have been purified and characterized (14,16,17,19,22,29,34). They have been divided in different classes according to their substrate specificity (9), electrophoretic mobility on polyacrylamide gels (9, 36), and stereospecific action on 2-monochloropropionic acid (2-MCPA) (36). Four different types of dehalogenation of 2-MCPA can be recognized. Two of these are represented by enzymes that are active with only L-or D-2-MCPA, giving products with inverted configuration at the chiral carbon atom. The other two act on both isomers, one with inversion of configuration and the other with retention of configuration.Previous studies have shown the presence of more than one haloacid dehalogenase in the same organism (9, 16), with only minor differences in substrate specificities. It has been suggested that these isoenzymes have arisen by gene duplication and subsequent divergent evolution (9).We are interested in the relation between structure, enzymatic mechanism, and evolution of dehalogenases. To study these aspects, we started to investigate the haloacid dehalogenase of Xanthobacter autotrophicus GJ1O. This bacterium was isolated on 1,2-dichloroethane as the sole carbon and energy source (13 In a previous paper, we reported the cloning of both dehalogenase genes from GJ10 (12). The haloalkane dehalogenase has been purified (15) and crystallized (25), and its sequence (12) and tertiary structure (5) have been determined. The haloacid dehalogenase gene has been cloned on a 10-kb fragment in the broad-host-range cosmid vector pLAFR1 (12). Here, we describe the purification and characterization of the haloacid dehalogenase from an overproducing mutant of GJ10 and the sequence of the gene encoding this enzyme. The properties of the enzyme are compared with those of other haloacid dehalogenases and haloalkane dehalogenase. MATERIALS AND METHODSGrowth conditions. Strains and plasmids used are listed in

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains ofAncylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhUA) genes of strains AD20 and AD25 were the same as the sequence of dhL4 from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.

* We would like to thank Tom Wansbeek, Ton Steerneman, Eric Zivot and anonymous referees for helpful comments. ABSTRACTThe paper considers the estimation of the coefficients of a single equation in the presence of dummy intruments. We derive pseudo ML and GMM estimators based on moment restrictions induced either by the structural form or by the reduced form of the model.The performance of the estimators is evaluated for the non-Gaussian case. We allow for heteroscedasticity. The asymptotic distributions are based on parameter sequences where the number of instruments increases at the same rate as the sample size. Relaxing the usual Gaussian assumption is shown to affect the normal asymptotic distributions. As a result also recently suggested new specification tests for the validity of instruments depend on Gaussianity. Monte Carlo simulations confirm the accuracy of the asymptotic approach.