Genetic characterization of the phenylacetyl-coenzyme A oxygenase from the aerobic phenylacetic acid degradation pathway of Escherichia coli

Abstract

We show here that the paaABCDE genes of the paa cluster responsible for phenylacetate degradation in Escherichia coli W encode a five-component oxygenase that hydroxylates phenylacetyl-coenzyme A (CoA), the first intermediate of the pathway. The primary structure of the subunits of bacterial phenylacetyl-CoA oxygenases revealed that these enzymes constitute the prototype of a new and distinct group of the large bacterial diiron multicomponent oxygenase family.

FIG. 1.

Genetic constructions used in this work. (A) Maps of plasmids pAFK3 and pFBP and their derivatives. (B) Maps of plasmids pAFBCK and pAFBK2. (C) Maps of plasmids pAFE, pAAD, and pAAD-ΔE. Arrows indicate the direction of gene transcription. Plasmid pAAD-ΔE was constructed by XmnI (X) digestion and religation of plasmid pAAD::Tn1000-83 (5), which eliminates most of the Tn1000 transposon and part of the paaE gene. Plasmids pAAD, pCK01, pUC18, and pAFK3 (a pSJ19Not derivative) have been previously described (5). Plasmids of the pAFD(A-E) series, pFBP, and pFBE are pUC18 derivatives. Plasmids of the pAF(A-E)K series are pAFK3 derivatives. Plasmids pAFBCK and pAFBK2 are pCK01 derivatives. Abbreviations: Ap^r, ampicillin resistance; Cm^r, chloramphenicol resistance. Plac, lac promoter. Pa, Px, and Pz are the promoters of the E. coli paa cluster.

FIG. 2.

Phylogenetic trees of the α subunits of BMOs. The bar represents 1 inferred amino acid substitution per 10 amino acids. Phylogenetic analyses were carried out according to the two-parameter method of Kimura (10), and a tree was reconstructed using the neighbor-joining method of the Phylip program (3). (A) Phylogenetic tree of the α subunits of representative members of different groups of BMOs. A schematic representation of the gene arrangement for the different groups of BMOs is also shown. Genes encoding the α, β, and γ subunits of the oxygenase component are indicated with black blocks. The genes encoding the reductase component (N) are indicated with stippled blocks. The genes encoding the effector protein (R) are indicated with hatched blocks. The genes encoding the Fp and T proteins in BMOs of groups 1 and 2, respectively, are shown with empty blocks. The amino acid lengths of the subunits are indicated in brackets below the corresponding proteins. The gene clusters shown are the following (GenBank accession numbers are in parentheses): dmpKLMNOP from Pseudomonas sp. strain CF600 (M60276), touABCDEF from P. stutzeri OX1 (AY621080), mmoXYBZorfYmmoC from Methylococcus capsulatus (M90050), amoABCD from Nocardia coralline B-276 (D37875), thmADBCorfQ from Pseudonocardia sp. strain K1 (AJ296087), and paaABCDE from E. coli W (X97452). The α subunits used for the construction of the phylogenetic tree are the following: DmpN (P19732) and TomA3 (AAL50373) of the phenol monooxygenase and toluene 2-monooxygenase from Pseudomonas sp. strain CF600 and Burkholderia cepacia G4, respectively; TouA (CAA06654) and TmoA (AAS66660) of the toluene/o-xylene monooxygenase and the toluene 4-monooxygenase from P. stutzeri OX1 and P. mendocina KR1, respectively; MmoX (AAB62392), of the methane monooxygenase from M. capsulatus; AmoC (BAA07114) of the alkene monooxygenase from N. coralline B-276; ThmA (CAC10506) of the tetrahydrofuran monooxygenase from Pseudonocardia sp. strain K1; and PaaA (CAA66090) of the phenylacetyl-CoA oxygenase from E. coli W. (B) Phylogenetic tree of the PaaA subunits of phenylacetyl-CoA oxygenases from E. coli (CAA66090), P. putida (AAC24334), Bordetella pertussis (NP_881296), Azoarcus evansii (AAG28968), Dechloromonas aromatica (YP_283606), Ralstonia eutropha (YP_297410), Rhodopseudomonas palustris (NP_949105), Sinorhizobium meliloti (CAC49959), Silicibacter pomeroyi (YP_283606), Bacillus halodurans (NP_241062), Geobacillus kaustophilus (YP_147897), Streptomyces coelicolor (NP_631518), Corynebacterium efficiens (NP_737277), Nocardia farcinica (YP_118371), Thermus thermophilus (YP_004583), and Deinococcus radiodurans (AAF11931).