trans-Dienelactone hydrolase from Pseudomonas reinekei MT1, a novel zinc-dependent hydrolase

Department of Microbial Pathogenesis, HZI-Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 10/2008; 376(2):423-8. DOI: 10.1016/j.bbrc.2008.09.006
Source: PubMed


Pseudomonas reinekei MT1 is capable of growing on 4- and 5-chlorosalicylate, involving a pathway with trans-dienelactone hydrolase (trans-DLH) as a key enzyme. It acts on 4-chloromuconolactone formed during cycloisomerization of 3-chloromuconate by hydrolyzing it to maleylacetate. The gene encoding this activity was localized, sequenced and expressed in Escherichia coli. Inductively coupled plasma mass spectrometry showed that both the wild-type as well as recombinant enzymes contained 2 moles of zinc but variable amounts of manganese/mol of protein subunit. The inactive metal-free apoenzyme could be reactivated by Zn(2+) or Mn(2+). Thus, trans-DLH is a Zn(2+)-dependent hydrolase using halosubstituted muconolactones and trans-dienelactone as substrates, where Mn(2+) can substitute for Zn(2+). It is the first member of COG1878 and PF04199 for which a direct physiological function has been reported.

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Available from: Howard Junca, Feb 28, 2014
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    • "The observed lack of inhibition of activity of purified TfdEI and TfdEII in the presence of EDTA in this study is similar to that reported for DLH from B. cepacia (previous name: Pseudomonas cepacia) [3] and Pseudomonas putida strain 87 [33]. On the contrary, the activity of the trans-dienelactone hydrolase from Pseudomonas reinekei MT1 is significantly inhibited by EDTA [4], and the enzyme has been recently reported to be zinc-dependent [10]. Surprisingly, both TfdEI and TfdEII do not hydrolyse substrate p-nitrophenyl (PNP) caprylate (C8) which suggests the absence of carboxylesterase activity whereas previous study on the hydrolysis of p-nitrophenyl (PNP) caprylate (C8) shows that some dienelactone hydrolases exhibit carboxylesterase activity [15]. "
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    ABSTRACT: In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (Mr 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs-1, with a Km value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs-1 and 0.0766 µMs-1, with a Km value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII kcat/Km ratios were 0.12 µM-1s-1and 0.13 µM-1s-1 and 0.216 µM-1s-1 and 0.094 µM-1s-1 for for cis- and trans-dienelactone, respectively. The kcat/Km ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though Km value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "This is the first time that the enzyme has been directly identified as a protein spot in a plant tissue. Carboxymethylenebutenolidase is an esterase involved in the degradation of aromatic compounds, it is poorly described in eukaryotes, while it has been described as a zinc dependent hydrolase in Pseudomonas reinekei [87]. "
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    ABSTRACT: Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with the roots of most plant species. While receiving photosynthates, they improve the mineral nutrition of the plant and can also increase its tolerance towards some pollutants, like heavy metals. Although the fungal symbionts exclusively colonize the plant roots, some plant responses can be systemic. Therefore, in this work a clone of Populus alba L., previously selected for its tolerance to copper and zinc, was used to investigate the effects of the symbiosis with the AM fungus Glomus intraradices on the leaf protein expression. Poplar leaf samples were collected from plants maintained in a glasshouse on polluted (copper and zinc contaminated) or unpolluted soil, after four, six and sixteen months of growth. For each harvest, about 450 proteins were reproducibly separated on 2DE maps. At the first harvest the most relevant effect on protein modulation was exerted by the AM fungi, at the second one by the metals, and at the last one by both treatments. This work demonstrates how importantly the time of sampling affects the proteome responses in perennial plants. In addition, it underlines the ability of a proteomic approach, targeted on protein identification, to depict changes in a specific pattern of protein expression, while being still far from elucidating the biological function of each protein.
    Full-text · Article · Jun 2012 · PLoS ONE
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    • "Site-directed mutagenesis. The previously constructed plasmid pASKtdlh [6] was used as template to introduce site-directed mutations in the trans-DLH encoding ccaC gene. Mutations were generated using the QuikChange "
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    ABSTRACT: Pseudomonasreinekei MT1 is capable of growing on 4- and 5-chlorosalicylate as the sole carbon source involving a pathway with trans-dienelactone hydrolase as the key enzyme. This enzyme transforms 4-chloromuconolactone to maleylacetate and thereby avoids the spontaneous formation of toxic protoanemonin. trans-Dienelactone hydrolase is a Zn(2+)-dependent hydrolase where activity can be modulated by the exchange of Zn(2+) by Mn(2+) in at least two of the three metal-binding sites. Site directed variants of conserved residues of the Q(101)XXXQ(105)XD(107)XXXH(111) motif and of H281 and E294 exhibit a two order of magnitude decrease in activity and a strong decrease in metal-binding capability. As none of the variants exhibited a change in secondary structure, the analyzed amino acid residues can be assumed to be involved in metal binding, forming a novel trinuclear metal-binding motif.
    Preview · Article · Nov 2009 · Biochemical and Biophysical Research Communications
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