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D J Hardman

Publications and source records attributed to D J Hardman.

14 recordsLinked to original sources
PubMed1998

Biochemical characterization of a haloalcohol dehalogenase from Arthrobacter erithii H10a.

Arthrobacter erithii H10a possesses two enzymes capable of catalyzing the dehalogenation of vicinal halohydrins which have been designated as dehalogenases DehA and DehC. The DehA dehalogenase demonstrated greater activity toward 1,3-dichloro-2-propanol (1,3-DCP) while the DehC dehalogenase showed higher activity toward 3-chloro-1,2-propanediol (3-CPD) and brominated alcohols. The DehA dehalogenase was composed of two non-identical subunits (relative molecular mass of 31.5 and 34 kDa) which probably associate with other proteins to form a large catalytically active protein of 200 kDa. The two subunits were purified and the amino acid sequence of their tryptic digests determined. The DehA enzyme catalyzed the conversion of vicinal halohydrins to epoxides and the reverse reaction in the presence of an excess of halogen. This enzyme had maximum activity at 50 degrees C and a broad pH optimum over the range 8.5-10.5. The apparent K(m) and Vmax values for dehalogenation of 1,3-DCP and 3-CPD were 0.105 mM and 223 mumol min-1 mg-1; and 2.366 mM and 1.742 mumol min-1 mg-1, respectively. The enzyme was inhibited by 2-chloroacetic acid (MCA) and 2,2-dichloroacetic acid (DCA). The inhibition pattern suggested a mixed type inhibition which was predominantly uncompetitive. Amino acid modification experiments demonstrated that one or more cysteine and arginine residues are likely to be involved in catalysis or play an important role in the maintenance of the enzyme structure. The characteristics of the DehA enzyme are compared to those of previously reported haloalcohol dehalogenases and discussed in terms of diversity of this type of dehalogenase.

Alcohols↗
PubMed1997

Some biochemical properties and the classification of a range of bacterial haloalkane dehalogenases.

Multivariate analyses and experimental data have been used to evaluate the relationships between eight bacterial hydrolytic haloalkane dehalogenases. The results indicate that seven of the dehalogenases investigated can confidently be placed into two Classes [sensu Slater, Bull and Hardman (1995) Biodegradation 6, 181-189] according to their substrate profiles. The remaining enzyme, isolated from Rhodococcus erythropolis CP9, appears to represent a third Class of haloalkane dehalogenases.

Amino Acid Sequence↗
PubMed1996

Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria.

The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated. Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles. Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein-1) and also had the highest affinity for 1,3-DCP (Km, 0.1 mM). Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (kl) of 1.13 h-1. The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations. However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl-1.

Biodegradation, Environmental↗
PubMed1995

Dehalogenation of haloalkanes by Rhodococcus erythropolis Y2. The presence of an oxygenase-type dehalogenase activity complements that of an halidohydrolase activity.

Rhodococcus erythropolis Y2 produced two types of dehalogenase: a hydrolytic enzyme, that is an halidohydrolase, which was induced by C3 to C6 1-haloalkane substrates, and at least one oxygenase-type dehalogenase induced by C7 to C16 1-haloalkanes and n-alkanes. The oxygenase-type activity dehalogenated C4 to C18 1-chloroalkanes with an optimum activity towards 1-chlorotetradecane. The halidohydrolase catalysed the dehalogenation of a wide range of 1- and alpha,omega-disubstituted haloalkanes and alpha,omega-substituted haloalcohols. In resting cell suspensions of hexadecane-grown R. erythropolis Y2 the oxygenase-type dehalogenase had a specific activity of 12.9 mU (mg protein)-1 towards 1-chlorotetradecane (3.67 mU mg-1 towards 1-chlorobutane) whereas the halidohydrolase in 1-chlorobutane-grown batch cultures had a specific activity of 44 mU (mg protein)-1 towards 1-chlorobutane. The significance of the two dehalogenase systems in a single bacterial strain is discussed in terms of their contribution to the overall catabolic potential of the organism.

Alkanes↗
PubMed1993

Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4.

The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme. These results, together with DNA sequence and analyses [Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem. J. 284, 87-93] provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase. It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.

Arginine↗
PubMed1992

Molecular biology of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4.

The structural gene (hdl IVa) for the Pseudomonas cepacia MBA4 2-haloacid halidohydrolase IVa (Hdl IVa) was isolated on a 1.6 kb fragment of Ps. cepacia MBA4 chromosomal DNA. The recombinant halidohydrolase was expressed in Escherichia coli and Pseudomonas putida and the structural gene was subcloned on to the tac expression vector pBTac1. High-level expression from the tac promoter was seen to be temperature-dependent, a consequence of the nucleotide sequence adjacent to the fragment encoding the halidohydrolase. The nucleotide sequence of the fragment encoding the Hdl IVa was determined and analysed. Three ATG codons were identified in one of the open reading frames and the one corresponding to the start of the hdl IVa structural gene was determined by comparison of the predicted amino acid sequences with the experimentally determined N-terminal sequences of halidohydrolase IVa. The hdl IVa gene encoded a 231-amino acid-residue protein of M(r) 25,900. The sequence and predicted structural data are discussed and comparison is made with sequence data for other halidohydrolases.

Amino Acid Sequence↗
PubMed1991

Biotransformation of halogenated compounds.

As a result of natural production and contamination of the environment by xenobiotic compounds, halogenated substances are widely distributed in the biosphere. Concern arises as a result of the toxic, carcinogenic, and potential teratogenic nature of these substances. The biotransformations of such halogenated substances are reviewed, with particular emphasis on the biocatalytic cleavage of the carbon-halogen bonds. The physiology, biochemistry, and genetics of the biological system involved in the dehalogenation reactions are discussed for three groups of organohalogens: (1) the haloacids, (2) the haloaromatics, and (3) the haloalkanes. Finally, the biotechnological applications of these microbial transformations are discussed. This includes prospects for their future application in biosynthetic processes for the synthesis of halogenated intermediates or novel compounds and also the use of such systems for the detoxification and degradation of environmental pollutants.

Bacteria↗
PubMed1990

Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2.

Rhodococcus erythropolis strain Y2, isolated from soil by enrichment culture using 1-chlorobutane, was able to utilize a range of halogenated aliphatic compounds as sole sources of carbon and energy. The ability to utilize 1-chlorobutane was conferred by a single halidohydrolase-type haloalkane dehalogenase. The presence of the single enzyme in cell-free extracts was demonstrated by activity strain polyacrylamide gel electrophoresis. The purified enzyme was a monomeric protein with a relative molecular mass of 34 kDa and demonstrated activity against a broad range of haloalkanes, haloalcohols and haloethers. The highest activity was found towards alpha, omega disubstituted chloro- and bromo- C2-C6 alkanes and 4-chlorobutanol. The Km value of the enzyme for 1-chlorobutane was 0.26 mM. A comparison of the R. erythropolis Y2 haloalkane halidohydrolase with other haloalkane dehalogenases is discussed on the basis of biochemical properties and N-terminal amino acid sequence data.

Chemical Phenomena↗
PubMed1986

Methods for isolating large bacterial plasmids.

In recent years, increasingly large plasmids have been isolated. This has been the result of improved methods for purifying large plasmids, some of which are reviewed in this article.

Bacteria↗