Research Areas
 |
|
Dehalogenases or halidohydrolases are hydrolytic enzymes which cleave the halogen-carbon bond(s) in halogenated aliphatic acids, yielding hydroxy- or oxo- alkanoic acids from mono- or disubstituted substrate, respectively. Dehalogenase producing microorganisms are normally selected for their ability to utilise halogenated alkanoic acids or haloalkanes as carbon and energy sources. Many microorganisms, however, possess dehalogenases for helping themselves to survive in hostile environment because many halogenated compounds are toxic.
Burkholderia cepacia MBA4 was isolated from batch culture using monobromoacetic acid as the sole carbon and energy source. This bacterium produced a dehalogenase (DehIVa) in batch culture condition. Previous studies on DehIVa using SDS-PAGE has suggested a molecular size of 23 kDa, whilst gel filtration indicated a molecular weight of about 45 kDa. This suggested that the active protein may be a dimer. The gene encoding for DehIVa has been cloned and sequenced. Analysis of the DNA sequence revealed an open reading frame for a protein of 231 amino acids and a molecular size of 25.9 kDa.
DehIVa and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Albeit their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes deh4a and dehCI. When amino acids 73-89 of DehCI was replaced by amino acids 74-90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a non-denaturing activity-stained gel. Similarly, when residues 73-89 of DehIVa were substituted by the corresponding residues of DehCI, the chimera migrated as a monomer. These seventeen amino acids changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis on deh4a however indicated that amino acids Phe 58, Thr 65, Leu 78 and Phe 92 of DehIVa are also important for the aggregation state of the protein. These indicate that the seventeen residues are not sufficient for the dimerization of the protein.
Amino and carboxyl terminal deletion derivatives of DehIVa were constructed and analyzed for enzyme activity and for protein integrity. The results suggested that the majority of the protein is indispensable. Point mutations on 29 conserved charged and/or polar residues were generated and characterized. Derivatives D11E, D11N, D11S and D181N were totally inactive while mutant N178D was defective in catalysis. Mutations of other conserved residues displayed varying effects. Mutation that enhances DehIVa activity has been shown to be inhibitory in other dehalogenase and essential conserved residues in DehIVa have been shown to be dispensable in others. This suggests there is no general rule for the importance of these conserved residues in the dehalogenases isolated so far.
Dehalogenase associated permease has been proposed to mediate the uptake of haloacid into the cell. We have cloned and expressed such a haloacid-specific transporter gene. The structural gene, designated as deh4p, was found located downstream of the coding sequence of DehIVa. The nucleotide sequence of deh4p was determined and characterized. An open reading frame of 1,656 bp encoding for a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. The nucleotide sequence of deh4p did not show any significant homology with any genes in the standard databases. A similar comparison with the assembled sequences of B. cepacia J2315, however, identified a region that shows 74% identity. Comparison of the predicted amino acid sequence with the databases shows that Deh4p has the signatures of sugar transport proteins and is an integral membrane protein of the major facilitator superfamily. deh4p has been cloned and expressed in E. coli. E. coli cells expressing Deh4p are more sensitive to monochloroacetate (MCA) and transport MCA rapidly into the cell. We are now in the process of charcterising this transporter protein.
In today's world where synthetic chemicals have been widely manipulated the fate and influence of these compounds to the environment and to the health of the human race attract much attention. The role of dehalogenases in detoxification or degradation of some of these man-made compounds should be investigated especially when high mutation rates appeared to be under environmental control. In my research I aim to obtain information on the molecular structure of dehalogenases and the expression of dehalogenases. The information obtained will shed light on the mechanism of the enzyme activity and helps engineering enzymes which can breakdown even more recalcitrant molecules or producing enzymes used in biotransformation procedure for chemical and biotechnological industries.
B. cepacia MBA4 has been shown to produce a single dehalogenase in batch culture condition. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture condition. We have clone and characterise one of the cryptic dehalogenases in MBA4. This cryptic dehalogenase, designated as Chd1, was expressed constitutively in E. coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as dimer. The subunits had relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards the L-isomer of 2-monochloropropionic acid only. The structural gene, chd1, was isolated on a 1.7-kb PstI fragment. This fragment contains a functional promoter because expression of chd1 in E. coli is orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding for a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence of chd1 did not show any homology with that of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42% to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence containing property of periplasmic enzymes.
Western blot analyses showed that Chd1 expressed in E. coli is translocated to the periplasm. The results on the expression of Chd1 in the presence of sodium azide suggested the cleavage of the leader to be Sec-dependent. Chimeras of Chd1 and green fluorescent protein demonstrated that the leader sequence is fully functional in translocating the fusion protein to the periplasm. The expression of the chimeras in Sec-mutants supported the Sec-dependent translocation. Surprisingly, recombinant Chd1 and a chimera with no leader sequence were also found in the periplasm. We are now in the process of characterising the properties of the Chd1 enzyme.
Dehalogenase genes isolated so far are mostly obtained from microorganisms capable of metabolising chlorinated or brominated compounds. Fluoroacetate dehalogenase has been isolated but it has not been studied thoroughly. This is in part due to the difficulty of obtaining fluoroacetate degrading microbes. Unlike chloro- and bromo-acetate, which have been used relatively successful in enriching for corresponding degradative bacteria, fluoroacetate is much more toxic to most microbes. This may be due to the stability of the carbon-fluorine bond and in part to the high electronegativity of the fluorine ion.
Conventional method in enrichment for dehalogenase possessing microbes used batch culture condition. This confers no problem for chloro- and bromo- compounds but this is not very successful in isolating fluoro-compound utilising bacteria. I will try to isolate fluoroacetate degrading microorganisms, as a start, using continuous culture method. The initial medium used for selection will only contain a small amount of fluoroacetate and supplemented with sufficient amount of utilisable carbon source such as pyruvate for growth. The condition will be controlled in such a way that a stable population can be maintained with the concentration of fluoroacetate increased gradually and with the supply of pyruvate decreased concomitantly. The culture can be checked for dehalogenase activity by the use of activity-stained polyacrylamide gel electrophoresis and by checking for the release of fluoride into the medium. Pure culture(s) obtained will then be selected for further study. The corresponding enzymes will be purified and characterised biochemically and the genes corresponding for the dehalogenases will be cloned by the use of molecular biology techniques. The physiology of the isolated microbes will also be investigated in order to understand the adaptation of the microbes in conditions where halogenated substrates are relatively abundant. Other fluorinated aliphatic acids will also be used for selection when the progress is achieved.
Halogenated aliphatic acids had been used as pesticides while haloalkanes had been used as organic solvents for dry-cleaning industry. The study of the degradation of haloalkanes is more difficult than the study of the haloaliphatics because the former compounds are normally insoluble and hence more difficult to handle, especially in making solid media. The alkanes are organic solvents therefore normal disposable plasticwares cannot be used. The handling of haloalkanes is therefore more labour intensive than usual. Special organic solvent-resistant labwares also have to be used for these volatile chemicals.
Factors which govern the dehalogenation mechanisms of halo-aliphatics also applied to the degradation of haloalkanes. Dehalogenases with activity towards 1-substituted alkanes are inactive towards 2-substituted derivatives. I would like to continue my previous projects on biodegradation of haloalkanes and broaden the understanding of microbial mineralisation of halogenated compounds.
Almost all of the currently studied dehalogenases for haloalkanoic acids and haloalkanes are of bacterial sources. This is probably in part due to the relatively ease of isolating and handling of unicellular organisms. Fungal metabolism of halogenated compounds by hydrolytic dehalogenases has not been studied. Eukaryotic microorganisms also provide another means of dehalogenation mechanism, namely, by oxygenation. This additional detoxification mechanism makes the isolation of hydrolytic dehalogenase containing eukaryotic microbes more complicated. I would like to include in my research the isolation and characterisation of hydrolytic dehalogenase producing fungi in order to compare the dehalogenases in pro- and eukaryotes. Initial screening for halidohydrolase can be detected by activity-stained polyacrylamide gel electrophoresis.
Last modified on 13th November, 2007.
Please send suggestions
and comments to jshtsang@hkucc.hku.hk.