4-sulfomuconolactone hydrolases from Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2.
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.
Halak
Sad
S
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Basta
Tamara
T
Bürger
Sibylle
S
Contzen
Matthias
M
Wray
Victor
V
Pieper
Dietmar Helmut
DH
Stolz
Andreas
A
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001557:Benzenesulfonates / Q000737:chemistry / Q000378:metabolism*; D002396:Catechols / Q000737:chemistry / Q000378:metabolism*; D042621:Comamonadaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008298:Maleates / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D011753:Pyrones / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013447:Sulfites / Q000378:metabolism
D001426:Bacterial Proteins; D001557:Benzenesulfonates; D002396:Catechols; D062385:Hydroxybenzoates; D008298:Maleates; D011753:Pyrones; D013447:Sulfites; C011905:maleoylacetic acid; C009091:protocatechuic acid; C409182:4-sulfocatechol; C024153:alpha-pyrone-4,6-dicarboxylic acid; D006867:Hydrolases