Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes

Abstract

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.Protocatechuate (PCA) is one of the key intermediate metabolites in the microbial catabolic pathways for various aromatic compounds, including phthalates, hydroxybenzoates, and lignin-derived aromatic compounds such as vanillate and ferulate. It is known that the aromatic ring fission of PCA is catalyzed by one of the three distinct dioxygenases PCA 3,4-dioxygenase (26), PCA 4,5-dioxygenase (36, 41), and PCA 2,3-dioxygenase (7). In the PCA 3,4-cleavage pathway, PCA is converted into 2-carboxy-cis,cis-muconate by the reaction catalyzed by PCA 3,4-dioxygenase, and the catabolic pathway for its product (␤-ketoadipate pathway) has been reported in many bacteria (24,26). In the case of the PCA 4,5-cleavage pathway, PCA is cleaved by PCA 4,5-dioxygenase to yield 4-carboxy-2-hydroxymuconate-6-semialdehyde, and then the product is degraded to 2-pyrone-4,6-dicarboxylate, 4-oxalomesaconate, and 4-carboxy-4-hydroxy-2-oxoadipate before entering the Krebs cycle (36). The genes and enzymes involved in this pathway have been recently characterized for several bacteria, such as Sphingobium (Sphingomonas) (36), Comamonas (47), Pseudomonas (35), and Arthrobacter (13) strains. On the other hand, no genetic information on the PCA 2,3-cleavage pathway has been reported since the finding of this pathway in some bacilli (7,8).In 1979, Crawford et al. reported the PCA 2,3-cleavage pathway of a 4-hydroxybenzoate (4HB) degrader, Paenibacillus sp. ...

Construction and Optimization of a Heterologous Pathway for Protocatechuate Catabolism in Escherichia coli Enables Bioconversion of Model Aromatic Compounds

“…This enzymatic activity can be provided by PraI from Paenibacillus sp. strain JJ-1B (16). We introduced the corresponding gene into the AG978 parental strain and the JME17 engineered strain, yielding strains JME7 and JME50, respectively (Fig.…”

“…The 2,3-cleavage pathway has been studied in Paenibacillus sp. strain JJ-1b, and heterologous expression of this pathway in E. coli enabled colony formation after a 10-day incubation (16). Finally, the most widely distributed pathway begins through 3,4-cleavage, as typified by Pseudomonas putida (13,17) (Fig.…”

Biodegradation of 3-Nitrobenzoate by Bacillus flexus strain XJU-4

“…and other Gram-positive organisms preferentially use 2,3-dioxygenase whereas Pseudomonas sp. and other Gram-negative organisms use 4,5-dioxygenase for the meta-cleavage of protocatechuate (Crawford et al 1979;Dennis et al 1973;Kasai et al 2009;Nadeau and Spain 1995;Urszula et al 2009;Wolgel and Lipscomb 1990). The degradation of 3-nitrobenzoate occurred by oxidative mechanism in both of these organisms whereas the degradation of other two isomers 2-nitrobenzoate and 4-nitrobenzoate have been shown to occur by reductive mechanism in Burkholderia terrae Strain KU-15 (Iwaki and Hasegawa 2007), Pseudomonas pickettii YH105 (Yabannavar and Zylstra 1995) and Comamonas acidovorans NBA-10 ( Groenewegen and de Bont 1992).…”

“…Data values represent the means of triplicates and error bars indicates 95% confidence intervals reported for 2-hydroxymuconic semialdehyde (Crawford 1975). This is a decarboxylated product of 5-carboxy-2-hydroxymuconic semialdehyde formed by 2,3-dioxygenase ring cleavage of protocatechuate by the enzymes in the crude extract (Crawford et al 1979;Wolgel and Lipscomb 1990;Kasai et al 2009). The activity of protocatechuate 2,3-dioygenase (specific activity 0.52 U/mg) was detected in 3-nitrobenzoate grown cell extracts but not in glucose grown cell extract, which suggest that the enzymes of 3-nitrobenzoate degradation were induced by growth of organism on 3-nitrobenzoate.…”

Transcriptional Regulation of the Terephthalate Catabolism Operon in Comamonas sp. Strain E6

“…The degradation of PCA is catalyzed by one of the following three aromatic ring cleavage pathways: the PCA 2,3-cleavage (17), the PCA 3,4-cleavage (14), or the PCA 4,5-cleavage (19,20) pathway. 2-Pyrone-4,6-dicarboxylate (PDC), which appears as an intermediate metabolite in the PCA 4,5-cleavage pathway (19,20), has been considered to be a useful chemical building block for the synthesis of biodegradable and high-function polymers (15,22,23).…”