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

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Abstract

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 Zn2+ or Mn2+. Thus, trans-DLH is a Zn2+-dependent hydrolase using halosubstituted muconolactones and trans-dienelactone as substrates, where Mn2+ can substitute for Zn2+. It is the first member of COG1878 and PF04199 for which a direct physiological function has been reported.

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Materials and methods

Bacterial strain and gene identification. Pseudomonas reinekei MT1 was grown and cell extracts prepared as previously described [12]. The gene fragment encoding the N-terminal protein sequence of trans-DLH was amplified by touchdown PCR (annealing temperatures of 60–50 °C) using primers YTNCCNACNTAYAAGCA and CCNGGNGGNGCRTCTTT, which were designed based on the previously determined N-terminal protein sequence (underlined) TDTSKSLPTYKQLLERKDAPPGSSWGLFGK [12]. A single 47 bp fragment was obtained,

Identification of the trans-DLH gene

The gene encoding trans-DLH involved in the degradation of 4- and 5-chlorosalicylate by P. reinekei MT1 was identified. The deduced N-terminus (TDTSKSLPTYKQLLERKDAPPGSSWGLFGK) of the identified gene was identical to the N-terminus previously described [12]. The gene comprises 1011 bp and encodes a protein of 336 amino acids, with a calculated molecular mass of 36.935 kDa. The deduced protein did not show similarity with any protein of assigned function described in public databases. Similarity

Discussion

A novel type of hydrolase, which is involved in the degradation of chlorosalicylates [12] has been characterized from P. reinekei MT1. PSI-BLAST searches using default parameters starting with the trans-DLH protein converged to about 300 proteins, many of which were annotated as putative cyclases or putative metal dependent hydrolases (see Supplementary Material). Conserved Domain Database [19] searches suggested that these proteins form a family that is classified as COG1878 in the Clusters of

Acknowledgments

This work was supported by the DFG-European Graduate College 653. We thank Iris Plumeier for excellent technical support. We are indebted to Dieter Zachmann from TU-Braunschweig and Manuel Ferrer from CSIC (Madrid) for their precious collaboration in the ICP-MS analysis and to Melissa Wos for critical reading of the manuscript.

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