Pseudomonas Putida PP3 Research Articles

English

Haloalkanoic acid dehalogenases remove halides from organic haloacids and have potential as bioremediation agents. DehE from Rhizobium sp. RC1, DehI from Pseudomonas putida PP3 and D,LDEX 113 from Pseudomonas sp. 113 are non-stereospecific dehalogenases that invert the configurations of D- and L- carbons bound to a halogen. The kinetics of DehE has been partially characterized and brominated compounds have greater specificity constant values than do the corresponding chlorinated compounds. The sequence of DehE is similar to that of DehI; therefore, the two enzymes may have similar structures and functions. The three-dimensional structure of DehI is known and its reaction mechanism was inferred from its structure and a mutagenesis study of D,L-DEX 113. Aspartate residues at positions 189 and 194 in DehI and D,L-DEX 113 were predicted to be involved in catalysis. These residues activate a water molecule that directly attacks the chiral carbon. Because DehE and DehI are sequentially related, delineating the structure of DehE is important to ascertain if the catalytic residues and reaction mechanism are the same for both enzymes. A structural prediction, sequence-homology modeling and a site-directed mutagenesis study of DehE might help achieve this goal. Key words: Haloalkanoic acids, non-stereospecific dehalogenase, DehE, Rhizobium sp. RC1, enzyme kinetics, protein structure prediction, site-directed mutagenesis.

Molecular Modelling and Functional Studies of the Non-Stereospecific α-Haloalkanoic Acid Dehalogenase (DehE) from Rhizobium SP. RC1 and its Association with 3-Chloropropionic Acid (β-Chlorinated Aliphatic Acid)

ABSTRACTMany environmental pollutions are caused by the abundance of xenobiotic compounds in nature. For instance, halogenated compounds released from chemical industries were proven to be toxic and recalcitrant in the environment. However, haloalkanoic acid dehalogenases can catalyse the removal of halides from organic haloacids and thus have gained interest for bioremediation and synthesis of industrial chemicals. This study presents the first structural model and the key residues of the non-stereospecific haloalkanoic acid dehalogenase, DehE, from Rhizobium sp. RC1. The enzyme was built using a homology modelling technique; the structure of DehI from Pseudomonas putida PP3 was used as a template, because of its homology to DehE. The structure of DehE consists of only α-helices. Twelve conserved residues that line the active site were identified: Trp34, Ala36, Phe37, Asn114, Tyr117 Ala187, Ser188, Asp189, Tyr265, Phe268, Ile269, and Ile272. These residues are consistent with the residues found in the active site of DehI and D, L-DEX 113 from Pseudomonas sp. 113. Asp189 activates the water molecule as a nucleophile to attack the substrate chiral centre, which would result in an inversion of configuration of either D- or L-substrates. Both D- and L-substrates bind to and interact with the enzyme by hydrogen bonding with three residues, Trp34, Phe37, and Ser188. In addition, a putative tunnel was also identified that would provide a channel for the substrate to access the binding site. Based on computational analysis, DehE was proven to have the substrate affinity towards 3-chloropropionic acid (3CP)/β-chlorinated aliphatic acid, however, its dehalogenation process is far from clear. This DehE structural information will allow for rational design of non-stereospecific haloalkanoic acid dehalogenases in the future.

Structure Prediction, Molecular Dynamics Simulation and Docking Studies of D-Specific Dehalogenase from Rhizobium sp. RC1

Currently, there is no three-dimensional structure of D-specific dehalogenase (DehD) in the protein database. We modeled DehD using ab initio technique, performed molecular dynamics (MD) simulation and docking of D-2-chloropropionate (D-2CP), D-2-bromopropionate (D-2BP), monochloroacetate (MCA), monobromoacetate (MBA), 2,2-dichloropropionate (2,2-DCP), D,L-2,3-dichloropropionate (D,L-2,3-DCP), and 3-chloropropionate (3-CP) into the DehD active site. The sequences of DehD and D-2-haloacid dehalogenase (HadD) from Pseudomonas putida AJ1 have 15% sequence similarity. The model had 80% of the amino acid residues in the most favored region when compared to the crystal structure of DehI from Pseudomonas putida PP3. Docking analysis revealed that Arg107, Arg134 and Tyr135 interacted with D-2CP, and Glu20 activated the water molecule for hydrolytic dehalogenation. Single residue substitutions at 25–30 °C showed that polar residues of DehD were stable when substituted with nonpolar residues and showed a decrease in activity within the same temperature range. The molecular dynamics simulation of DehD and its variants showed that in R134A variant, Arg107 interacted with D-2CP, while in Y135A, Gln221 and Arg231 interacted with D-2CP. It is our emphatic belief that the new model will be useful for the rational design of DehDs with enhanced potentials.

A new dl-2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

dl-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of d- and l-2-haloalkanoic acids to produce the corresponding l- and d-2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for dl-2-haloacid dehalogenase from Pseudomonas putida PP3 ( dl-DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of dl-DEX 312 was carried out in H 2 18 O by use of a large excess of the enzyme with d- or l-2-chloropropionate as a substrate, the lactate produced was labeled with 18 O . This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of l-2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. dl-DEX 312 resembles dl-2-haloacid dehalogenase from Pseudomonas sp. 113 ( dl-DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, dl-DEX 312 is markedly different from dl-DEX 113 in its substrate specificity. We found that dl-DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for dl-DEX 113. dl-DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.

Cloning and nucleotide sequence of a D,L-haloalkanoic acid dehalogenase encoding gene from Alcaligenes xylosoxidans ssp. denitrificans ABIV

We have cloned DNA fragments of plasmid pFL40 from Alcaligenes xylosoxidans ssp. denitrificans ABIV encoding a D,L-2-haloalkanoic acid halidohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expression of the halidohydrolase was cloned in Pseudomonas fluorescens and Escherichia coli. A 1.9-kb HindII-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coli. The nucleotide sequence of this DNA-fragment was determined. It had an open reading frame coding for 296 amino acid residues (molecular weight of 32783 D). The dhlIV gene showed sequence homology to a short segment of a D-specific dehalogenase (hadD) from Pseudomonas putida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhlIV and the D,L- isomer specific dehl dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a sigma 54-dependent activator protein, similar to the dehRI regulatory gene from Pseudomonas putida PP3 is located upstream of dhlIV. In contrast to DehI, dehalogenation of D- or L-chloropropionic acid by the DhlIV-protein leads to lactic acid of inverted configuration.

The nucleotide sequence of a transposable haloalkanoic acid dehalogenase regulatory gene (dehR I) fromPseudomonas putida strain PP3 and its relationship with ?54-dependent activators

The mobile genetic element, DEH found in Pseudomonas putida PP3 carries a 2-haloalkanoic acid dehalogenase structural gene, dehI, and its associated regulatory gene, dehRI. The nucleotide sequence of dehRI was determined. The gene had an open reading frame putatively encoding for a 64 kDa protein containing 571 amino acid residues. The protein was similar to previously published sequences of several other sigma 54-dependent activator proteins. Amino acid sequence analysis showed that the deduced DehRI protein clustered with the NifA nitrogenase regulatory activator family, and was most closely related, with 47.7% similarity, to a 'NifA-like' deduced partial sequence from a plasmid-encoded ORF in Pseudomonas sp. strain NS671, associated with L-amino acid production. The domain structure of DehRI was analysed by alignment with other NifA-like and NtrC-like sequences and showed a highly conserved central region of approximately 230 amino acids, and a potential DNA-binding domain. No homology was detected between the deduced DehRI and other sigma 54-dependent activator sequences at the N-terminus, a result which was consistent with that region being the domain which recognised inducer.

The dehalogenase gene dehI from Pseudomonas putida PP3 is carried on an unusual mobile genetic element designated DEH.

As a result of the production of two dehalogenases (DehI and DehII), Pseudomonas putida PP3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2MCPA), as sole sources of carbon and energy. The DehI gene (dehI) was carried on a mobile genetic element (DEH) located on the chromosome of strain PP3. DEH recombined with target plasmid DNAs at high frequencies (e.g. 3.8 x 10(-4) per RP4.5 plasmid transferred). The regulated expression of dehI was detected in P. putida, Pseudomonas aeruginosa, and Escherichia coli strains containing derivative plasmids of RP4.5 and pWW0 recombined with DEH. Movement of DEH from the unstable RP4 derivatives pNJ5000 and pMR5 resulted in the insertion of DEH into the chromosome of RecA+ strains of P. putida but not in RecA+ nor RecA- strains of E. coli. Rescue of DEH from the chromosome of P. putida KT2441 onto plasmid RP4 involved recombination at a frequency (2.7 x 10(-4) per RP4 plasmid transferred) comparable to that observed in strain PP3. The DEH element was not classified as a conventional transposon because it did not move as a discrete DNA fragment: dehI-containing inserts in plasmid DNA targets varied in size between 6 and 13 kb. In addition, DEH exhibited a marked preference for insertion into a specific site on the plasmid pWW0, but its transposition, independent of host recombinational systems, remains to be demonstrated. However, the transposonlike characteristics of DEH included the conservation of restriction endonuclease sites, high-frequency recombination with different target replicons (plasmid and chromosomal DNA), and promiscuous insertion into plasmid RP4-based replicons. Therefore, it is proposed that DEH is an unusual mobile genetic element.

Localization and functional analysis of structural and regulatory dehalogenase genes carried on DEH from Pseudomonas putida PP3.

Pseudomonas putida PP3 expressed two dehalogenases, DehI and DehII. The DehI gene (dehI) was located on a mobile DNA element (DEH) which inserted at high frequencies into target plasmids from its chromosomal location. From a recombinant TOL plasmid (pWW0) containing a 6.0-kb DEH element inserted into the plasmid's 5.6-kb EcoRI-G restriction endonuclease fragment, an 11.6-kb EcoRI fragment was cloned. Subcloning analysis and insertion mutagenesis produced a structural map of the DEH element and located the dehalogenase functions. The gene dehI was transcribed from a regulated promoter on DEH which was expressed in P. putida and Escherichia coli. The direction of transcription of dehI was determined, and it was also found to be under positive control, activated by an adjacent regulatory gene (dehRI). Expression of dehI in clones containing the intact DEH supported good growth on 2-monochloropropionate (2MCPA). Subclones lacking dehRI expressed dehI at levels which allowed only slow growth on 2MCPA, even when dehI expression was initiated from vector promoters. Expression of dehI in P. putida containing the intact DEH element required rpoN, suggesting that it was omega 54 dependent. The intact DEH element transferred to P. putida on a suicide plasmid donor pAWT34 (pBR325 replicon), and dehI was stably inherited, without vector DNA sequences, in transformants selected on 2MCPA. This indicated that the cloned DEH element contained functions associated with recombination.

Dehalogenase genes of Pseudomonas putida PP3 on chromosomally located transposable elements.

Pseudomonas putida PP3 utilizes halogenated alkanoic acids (HAA) such as 2,2-DCPA as its sole carbon and energy sources. Spontaneous HHA- mutants, isolated by selection for resistance to the toxic analogs monochloroacetic acid and dichloroacetic acid, arose at frequencies several orders of magnitude higher than expected for spontaneous mutations. Analysis of the five classes of mutants isolated suggested that the dehalogenase and HAA permease genes were on chromosomally located transposable elements and that the spontaneous mutations involved excision of these elements. This suggestion was confirmed by the observation that one of the elements can transpose to a target DNA molecule. The frequency of the excision event was strongly influenced by environmental conditions. Possible relationships between expression of cryptic genes and their location on transposable elements are discussed.

Toxic effects of chlorinated and brominated alkanoic acids on Pseudomonas putida PP3: selection at high frequencies of mutations in genes encoding dehalogenases.

Mutant strains of Pseudomonas putida PP3 capable of utilizing monochloroacetate (MCA) and dichloroacetate (DCA) as the sole sources of carbon and energy were isolated from chemostat cultures. The mutants differed from the parent strain in that they could grow on products of MCA and DCA dehalogenation (catalyzed by inducible dehalogenases I and II) and were resistant to growth inhibition by the two substrates. The growth inhibition of strain PP3 by MCA, DCA, and other halogenated alkanoic acids was studied. Sensitivity to dehalogenase substrates was related to the expression of the dehalogenase genes. For example, mutants producing elevated levels of one or both of the dehalogenases were sensitive to 2-monochloropropionate and 2-monochlorobutanoate at concentrations which did not affect the growth of strain PP3. P. putida PP1, the parent of strain PP3, was resistant to the inhibitory effects of MCA and DCA. Spontaneous mutants of strain PP3, also resistant to MCA and DCA, were selected at high frequency, and four different classes of these strains were distinguished on the basis of dehalogenase phenotype. All dehalogenase-producing mutants were inducible; no constitutive mutant has yet been isolated. Most of the resistant mutants examined did not produce one or both of the dehalogenase, and over half of those tested failed to revert back to the parental (strain PP3) phenotype, indicating that the observed mutations involved high-frequency deletion of DNA base sequences affecting expression of genes encoding dehalogenases and associated permease(s).

The Formation of an R-prime Carrying the Fraction I Dehalogenase Gene from Pseudomonas putida PP3 using the IncP Plasmid R64.44

SUMMARY: Plasmid R68.44 was transferred to Pseudomonas putida PP3 at a frequency of approximately 10-6. In the new strain, P. putida PPW1, the plasmid, designated pUU1, lost a 2.0 kb fragment corresponding to IS21 which had previously been implicated in enhanced chromosome mobilization, but gained between 3.4 and 5.7 kb of DNA. Plasmid pUU1 was used to mobilize the fraction I dehalogenase gene at a low frequency of approximately 10-1 into another strain of P. putida. The R-prime plasmid, designated pUU2, contained an additional 9.2 to 11.0kb fragment which carried the fraction I dehalogenase.

Stereospecificity of 2-monochloropropionate dehalogenation by the two dehalogenases of Pseudomonas putida PP3: evidence for two different dehalogenation mechanisms.

Pseudomonas putida PP3 grew on DL-2-monochloropropionate (2MCPA) with a release of chloride ions consistent with the dechlorination of both isomers. The organism grew on either D- or L-2MCPA. Dehalogenase activity in cell-free extracts showed that both D- and L-2MCPA were dehalogenated. Pseudomonas putida PP3 contains two dehalogenases, and studies with the separated enzymes revealed that the fraction I enzyme used both D- and L-2MCPA, the rate of dechlorination of L-2MCPA being 80% of the rate of D-2MCPA dechlorination. The product of the reaction, lactate, retained the same optical configuration as the substrate provided. The fraction II dehalogenase also dechlorinated D- and L-2MCPA, with the same difference in rates as for the fraction I dehalogenase, but the lactates produced were of the opposite configuration to their precursors. The two dehalogenases showed further differences with respect to inhibition by two sulphydryl-blocking agents, N-ethylmaleimide and p-chloromercuribenzoate. Fraction I dehalogenase was considerably more sensitive to these two reagents compared with the fraction II dehalogenase. Dithiothreitol partially protected the fraction I dehalogenase from N-ethylmaleimide inhibition. The results are discussed in terms of the possible evolutionary relationships of the two dehalogenases.

Dehalogenases in Soil Bacteria

Sixteen bacterial strains isolated from soil were able to grow on either 2-monochloropropionic acid or monochloroacetic acid as the sole carbon and energy source. The isolates were divided into five groups on the basis of differences in their dehalogenase activities towards four substrates - monochloroacetic acid, dichloroacetic acid, 2-monochloropropionic acid and 2,2-dichloropropionic acid. Disc gel electrophoresis of crude extracts identified four distinct dehalogenases with different electrophoretic mobilities: three isolates contained one, three or four dehalogenases, respectively, and the remaining 13 isolates contained different combinations of two dehalogenases. In some cases, dehalogenases with the same mobilities from different isolates appeared to be identical enzymes. In others, enzymes from different isolates with the same electrophoretic mobility had different substrate activity profiles. Pseudomonas putida PP3 was shown to contain one enzyme which was comparable with one of the dehalogenases detected in several of the newly isolated soil bacteria. The second enzyme was not found in soil bacteria and represented a fifth dehalogenase. The significance of these results in terms of the evolution of dehalogenase activity is discussed.

The partial purification of two dehalogenases from Pseudomonas putida PP3

The partial purification of two dehalogenases from Pseudomonas putida PP3