Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes
Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes
复制标题
DOI：
10.1128/jb.00840-09
复制
发表时间：
2009-11-01
影响因子：
3.2
通讯作者：
Masai, Eiji
中科院分区：
文献类型：
--
作者：
Kasai, Daisuke;Fujinami, Toshihiro;Masai, Eiji
类芽孢杆菌（Paenibacillus sp.）（以前的浸软芽孢杆菌（Bacillus macerans））菌株JJ-1b能够在4-羟基苯甲酸（4 HB）上生长作为唯一的碳源和能量源，并且已知通过原儿茶酸（PCA）2，3-裂解途径降解4 HB。然而，没有一个参与这一途径的基因被确定。在这项研究中，我们确定并表征了JJ-1b基因的4 HB分解代谢途径通过PCA 2，3-裂解途径，其中包括praR和praABEGFDCHI。基于携带praI、praA、praH、praB、praC和praD的大肠杆菌细胞提取物的酶活性，发现这些基因分别编码4 HB 3-羟化酶、PCA 2，3-双加氧酶、5-羧基-2-羟基粘康酸-6-半醛脱羧酶、2-羟基粘康酸-6-半醛脱氢酶、4-羟巴豆酸（OCA）互变异构酶和OCA脱羧酶，其参与4 HB转化为2-羟基戊-2，4-二烯酸（HPD）。praE、praF和praG基因产物与负责将HPD催化转化为丙酮酸和乙酰辅酶A的相应酶具有45 - 61%的氨基酸序列同一性。推导的praR的氨基酸序列与IclR类转录调控因子的氨基酸序列相似。逆转录-PCR分析表明praABEGFDCHI构成一个操纵子，并且这些基因在JJ-1b在4 HB和PCA上的生长过程中表达。praR-praABEGFDCHI将在4 HB上生长的能力赋予E.表明praEGF对HPD转化为丙酮酸和乙酰辅酶A具有功能性。启动子分析表明，praR编码pra操纵子的阻遏物。
Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of praR and praABEGFDCHI. Based on the enzyme activities of cell extracts of Escherichia coli carrying praI, praA, praH, praB, praC, and praD, these genes were found to code for 4HB 3-hydroxylase, PCA 2,3-dioxygenase, 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate (OCA) tautomerase, and OCA decarboxylase, respectively, which are involved in the conversion of 4HB into 2-hydroxypenta-2,4-dienoate (HPD). The praE, praF, and praG gene products exhibited 45 to 61% amino acid sequence identity to the corresponding enzymes responsible for the catabolism of HPD to pyruvate and acetyl coenzyme A. The deduced amino acid sequence of praR showed similarity with those of IclR-type transcriptional regulators. Reverse transcription-PCR analysis revealed that praABEGFDCHI constitute an operon, and these genes were expressed during the growth of JJ-1b on 4HB and PCA. praR-praABEGFDCHI conferred the ability to grow on 4HB to E. coli, suggesting that praEGF were functional for the conversion of HPD to pyruvate and acetyl coenzyme A. A promoter analysis suggested that praR encodes a repressor of the pra operon.