Protocatechuate 4,5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases

Abstract

Comamonas testosteroni T-2 degraded at least eight aromatic compounds via protocatechuate (PCA), whose extradiol ring cleavage to 2-hydroxy-4-carboxymuconate semialdehyde (HCMS) was catalysed by PCA 4,5-dioxygenase (PmdAB). This inducible, heteromultimeric enzyme was purified. It contained two subunits, a (PmdA) and b (PmdB), and the molecular masses of the denatured proteins were 18 kDa and 31 kDa, respectively. PCA was converted stoichiometrically to HCMS with an apparent K m of 55 lM and at a maximum velocity of 1.5 lkat. Structureactivity-relationship analysis by testing 16 related compounds as substrate for purified PmdAB revealed an absolute requirement for the vicinal diol and for the carboxylate group of PCA. Besides PCA, only 5¢-hydroxy-PCA (gallate) induced oxygen uptake. The Nterminal amino acid sequence of each subunit was identical to the corresponding sequences in C. testosteroni BR6020, which facilitated sequencing of the pmdAB genes in strain T-2. Small differences in the amino acid sequence had significant effects on enzyme stability. Several homologues of pmdAB were found in sequence databases. Residues involved in substrate binding are highly conserved among the homologues. Their sequences grouped within the class III extradiol dioxygenases. Based on our biochemical and genetic analyses, we propose a new branch of the heteromultimeric enzymes within that class.

Bacterial degradation of phthalate isomers and their esters

“…1.13.11.8). Both these enzymes are well characterized and found to be heterodimers with α and β subunits and requires Fe +2 for their activity [32,64,67,76,104,109,111,112]. Some of them are reported to be octamers or decamers [5,25,105,108].…”

Functional Metagenomics of a Biostimulated Petroleum-Contaminated Soil Reveals an Extraordinary Diversity of Extradiol Dioxygenases

“…These results combined with the substrates utilized by the type II Edos that have been characterized so far suggest that at least some families (II.1 and II.6) of type II Edos may not be particularly active against catechol. On the other hand, the enzymes of the II.2 and II.5 families that have been characterized to date (not found in this screening), such as MhpB and McpI (4), GalA (6), DesB and LigAB (27), and PmdAB (46), are clearly involved in cleaving the aromatic rings of monocyclic substrates. This reinforces the view that the substrate preferences of Edos may be related to their phylogenetic origins (1,2).…”

Degradation of 4-chlorobiphenyl and 4-chlorobenzoic acid by the strain Rhodococcus ruber P25

“…Con cerning the catalytic characteristics, the isolated enzyme differs from the known PCA 4,5 DOs both in the substrate affinity and in the catalytic activity. The distinctive feature of the isolated enzyme is its rela tively high thermostability compared to the enzymes described in literature [17,33].…”

“…PCA catabolism may proceed by the meta pathway with formation of 2 hydroxy 4 car boxy muconic aldehyde; the reaction is performed by protochatechuate 4,5 dioxygenase. Such type of metabolism has been described for a number of gram negative bacteria, including Comamonas test osteroni T 2 [33] and Pseudomonas sp. [17].…”