Characterization of a sulfur-regulated oxygenative alkylsulfatase from Pseudomonas putida S-313
Characterization of a sulfur-regulated oxygenative alkylsulfatase from Pseudomonas putida S-313
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DOI：
10.1074/jbc.m005820200
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发表时间：
2000-10-13
影响因子：
4.8
通讯作者：
Kertesz, MA
中科院分区：
文献类型：
--
作者：
Kahnert, A;Kertesz, MA
恶臭假单胞菌S-313的atsK基因是以烷基硫酸酯为硫源生长所必需的。AtsK蛋白在大肠杆菌中过表达并纯化至均一。序列分析表明AtsK与E. coli牛磺酸双加氧酶（38%氨基酸同一性）。AtsK蛋白催化一系列烷基硫酸酯的α-酮戊二酸依赖性裂解，其链长范围为C-4至C-12，需要氧和Fe 2+才能发挥活性，并释放琥珀酸盐、硫酸盐和相应的醛作为产物。酶的活性是最佳的pH值为7，并强烈刺激抗坏血酸。与大多数其他表征的α-酮戊二酸依赖性双加氧酶不同，AtsK接受一系列α-酮酸作为共底物，包括α-酮戊二酸（K-m 140 μ M）、α-酮己二酸、α-酮戊酸和α-酮辛酸。硫酸己酯和SDS的实测Km值分别为40和34 μ M。纯化酶的表观M-r为121，000，与同源四聚体结构一致，这对于该酶超家族是不寻常的，其成员通常是单体或二聚体。AtsK酶的性质和氨基酸序列因此将其定义为不寻常的氧分解烷基硫酸酯酶和α-酮戊二酸依赖性双加氧酶家族的新成员。
The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid identity). The AtsK protein catalyzed the alpha-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C-4 to C-12, required oxygen and Fe2+ for activity and released succinate, sulfate, and the corresponding aldehyde as products. Enzyme activity was optimal at pH 7 and was strongly stimulated by ascorbate. Unlike most other characterized alpha-ketoglutarate-dependent dioxygenases, AtsK accepted a range of alpha-keto acids as co-substrates, including alpha-ketoglutarate (K-m 140 mu M), alpha-ketoadipate, alpha-ketovalerate, and alpha-ketooctanoate. The measured K-m values for hexyl sulfate and SDS were 40 and 34 mu M, respectively. The apparent M-r of the purified enzyme of 121,000 was consistent with a homotetrameric structure, which is unusual for this enzyme superfamily, members of which are usually monomeric or dimeric, The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytic alkylsulfatase and a novel member of the alpha-ketoglutarate-dependent dioxygenase family.