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Journal of Bacteriology, October 2007, p. 6998-7006, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00611-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

4-Sulfomuconolactone Hydrolases from Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

Sad Halak,¹ Tamara Basta,^1, Sibylle Bürger,¹ Matthias Contzen,^1, Victor Wray,² Dietmar Helmut Pieper,² and Andreas Stolz^1*

Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany,¹ Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany²

Received 20 April 2007/ Accepted 19 July 2007

The 4-carboxymethylen-4-sulfo-but-2-en-olide (4-sulfomuconolactone) hydrolases from Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain S2 are part of a modified protocatechuate pathway responsible for the degradation of 4-sulfocatechol. In both strains, the hydrolase-encoding genes occur downstream of those encoding the enzymes that catalyze the lactonization of 3-sulfomuconate. The deduced amino acid sequences of the 4-sulfomuconolactone hydrolases demonstrated the highest degree of sequence identity to 2-pyrone-4,6-dicarboxylate hydrolases, which take part in the meta cleavage pathway of protocatechuate. The 4-sulfomuconolactone hydrolases did not convert 2-pyrone-4,6-dicarboxylate, and the 2-pyrone-4,6-dicarboxylate hydrolase from Sphingomonas paucimobilis SYK-6 did not convert 4-sulfomuconolactone. Nevertheless, the presence of highly conserved histidine residues in the 4-sulfomuconolactone and the 2-pyrone-4,6-dicarboxylate hydrolases and some further sequence similarities suggested that both enzymes belong to the metallo-dependent hydrolases (the "amidohydrolase superfamily"). The 4-sulfomuconolactone hydrolases were heterologously expressed as His-tagged enzyme variants. Gel filtration experiments suggested that the enzymes are present as monomers in solution, with molecular weights of approximately 33,000 to 35,000. 4-Sulfomuconolactone was converted by sulfomuconolactone hydrolases to stoichiometric amounts of maleylacetate and sulfite. The 4-sulfomuconolactone hydrolases from both strains showed pH optima at pH 7 to 7.5 and rather similar catalytic constant (k_cat/K_M)values. The suggested 4-sulfocatechol pathway from 4-sulfocatechol to maleylacetate was confirmed by in situ nuclear magnetic resonance analysis using the recombinantly expressed enzymes.

Published ahead of print on 27 July 2007.

Present address: Institut Pasteur, 25 Rue du Dr. Roux, F-75015 Paris, France.

Present address: Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Germany.

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