Halak Sad, Basta Tamara, Bürger Sibylle, Contzen Matthias, Wray Victor, Pieper Dietmar Helmut, Stolz Andreas
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
J Bacteriol. 2007 Oct;189(19):6998-7006. doi: 10.1128/JB.00611-07. Epub 2007 Jul 27.
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.
中间氢噬菌S1菌株和放射形土壤杆菌S2菌株中的4-羧甲基-4-磺基-2-丁烯内酯（4-磺基粘康酸内酯）水解酶是负责降解4-磺基邻苯二酚的改良原儿茶酸途径的一部分。在这两种菌株中，水解酶编码基因位于催化3-磺基粘康酸内酯化的酶编码基因的下游。4-磺基粘康酸内酯水解酶的推导氨基酸序列与参与原儿茶酸间位裂解途径的2-吡喃酮-4,6-二羧酸水解酶具有最高程度的序列同一性。4-磺基粘康酸内酯水解酶不转化2-吡喃酮-4,6-二羧酸，少动鞘氨醇单胞菌SYK-6的2-吡喃酮-4,6-二羧酸水解酶也不转化4-磺基粘康酸内酯。然而，4-磺基粘康酸内酯和2-吡喃酮-4,6-二羧酸水解酶中高度保守的组氨酸残基的存在以及一些进一步的序列相似性表明这两种酶都属于金属依赖性水解酶（“酰胺水解酶超家族”）。4-磺基粘康酸内酯水解酶作为His标签酶变体进行异源表达。凝胶过滤实验表明，这些酶在溶液中以单体形式存在，分子量约为33,000至35,000。4-磺基粘康酸内酯被磺基粘康酸内酯水解酶转化为化学计量的马来酰乙酸和亚硫酸盐。两种菌株的4-磺基粘康酸内酯水解酶在pH 7至7.5时表现出最佳pH值，并且催化常数（k(cat)/K(M)）值相当相似。使用重组表达的酶进行的原位核磁共振分析证实了从4-磺基邻苯二酚到马来酰乙酸的推测4-磺基邻苯二酚途径。
