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 truns-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. (C) 2008 Elsevier Inc. All rights reserved.