Reversible and nonoxidative γ-resorcylic acid decarboxylase:: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter
Reversible and nonoxidative γ-resorcylic acid decarboxylase:: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter
复制标题
DOI：
10.1016/j.bbrc.2004.09.091
复制
发表时间：
2004-11-12
影响因子：
3.1
通讯作者：
Kirimura, K
中科院分区：
文献类型：
--
作者：
Ishii, Y;Narimatsu, Y;Kirimura, K
从γ-间苯二酚（RE，1，3-dihydroxybenzene）降解菌中筛选得到一株辐射根瘤菌（Rhizobium radiobacterium WU-0108），在该菌株中发现了一种γ-间苯二酚（γ-RA，2，6-dihydroxybenzoic acid）脱羧酶，该酶可用于间苯二酚（RE，1，3-dihydroxybenzene）羧化生成γ-RA。结果表明，在红曲霉无细胞提取物中检测到RE羧化和γ-RA脱羧活性。辐射藻WU-0108在有氧条件下与γ-RA一起生长。通过一次离子交换层析和两次疏水层析，在SDS-PAGE上将γ-RA脱羧酶纯化至均一。凝胶过滤法测得该酶的分子量为130 kDa，SDS-PAGE法测得其亚基的分子量为34 kDa，表明该酶为同源四聚体结构。该酶催化γ-RA的脱羧，但不催化α-RA或β-RA。在不添加任何辅因子的情况下，该酶催化RE的区域选择性羧化以形成γ-RA，而不形成α-RA和P-RA，以及邻苯二酚至2，3-二羟基苯甲酸。在有氧条件下，该酶的γ-RA脱羧活性和RE羧化活性均未降低，这与目前报道的其它脱羧酶不同。将编码γ-RA脱羧酶的基因rdc克隆到大肠杆菌中，测序，并进行过表达。推导的rdc基因的氨基酸序列由327个氨基酸残基组成，对应于34 kDa的蛋白质，并显示出42%和30%的同一性，从尼日尔曲霉和5-羧基香草酸脱羧酶从少动分裂单胞菌SYK-6的2，3-二羟基苯甲酸脱羧酶。一个定点突变的研究表明，两个组氨酸残基的位置164和2 - 18的Rdc是必不可少的γ-RA和RE的羧化的脱羧催化活性。(C)2004年爱思唯尔公司All rights reserved.
We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and P-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Splingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 2 18 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE. (C) 2004 Elsevier Inc. All rights reserved.