Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240
Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240
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DOI：
10.1007/s00203-006-0117-5
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发表时间：
2006-07-01
影响因子：
2.8
通讯作者：
Nagasawa, Toru
中科院分区：
文献类型：
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作者：
Matsui, Tsuyoshi;Yoshida, Toyokazu;Nagasawa, Toru
我们发现4-羟基苯甲酸脱羧酶发生在阴沟肠杆菌P240，在厌氧条件下从土壤中分离，并纯化酶的同质性。纯化的酶是相同的60 kDa亚基的homohexamer。纯化的脱羧酶催化4-羟基苯甲酸的非氧化脱羧，而不需要任何辅助因子。其对4-羟基苯甲酸酯的K（m）值为596 μ M。该酶还催化3，4-二羟基苯甲酸酯的脱羧反应，其K（m）值为6.80 mM。在3 M KHCO 3和20 mM苯酚存在下，该脱羧酶催化苯酚的逆羧化反应形成4-羟基苯甲酸酯，摩尔转化率为19%。对苯酚的K（m）值为14.8mM。Clopidin P240.重组质粒的核苷酸序列分析表明，4-羟基苯甲酸脱羧酶基因编码475个氨基酸的蛋白质。该酶的氨基酸序列与羟基苯甲酸梭菌（Clostridium hydroxybenzoicum）的4-羟基苯甲酸脱羧酶（53%同一性）、链霉菌属菌株D 7的VdcC蛋白（香草酸脱羧酶）（72%）和大肠杆菌的3-八异戊二烯基-4-羟基苯甲酸脱羧酶（28%）的氨基酸序列相似。这些假设的蛋白质与大肠杆菌的一级结构具有96-97%的同一性。在几种细菌菌株中发现了Clobacillus P240 4-羟基苯甲酸脱羧酶。
We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K (m) value for 4-hydroxybenzoate was 596 mu M. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K (m) value was 6.80 mM. In the presence of 3 M KHCO3 and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K (m) value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96-97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.