title: Molecular characterization of the alpha subunit of multicomponent phenol hydroxylase from 4-chlorophenol-degrading Pseudomonas sp. strain PT3.
abstract: Multicomponent phenol hydroxylases (mPHs) are diiron enzymes that use molecular oxygen to hydroxylate a variety of phenolic compounds. The DNA sequence of the alpha subunit (large subunit) of mPH from 4-chlorophenol (4-CP)-degrading bacterial strain PT3 was determined. Strain PT3 was isolated from oil-contaminated soil samples adjacent to automobile workshops and oil stations after enrichment and establishment of a chlorophenol-degrading consortium. Strain PT3 was identified as a member of Pseudomonas sp. based on sequence analysis of the 16S rRNA gene fragment. The 4-CP catabolic pathway by strain PT3 was tentatively proposed to proceed via a meta-cleavage pathway after hydroxylation to the corresponding chlorocatechol. This hypothesis was supported by polymerase chain reaction (PCR) detection of the LmPH encoding sequence and UV/VIS spectrophotometric analysis of the culture filtrate showing accumulation of 5-chloro-2-hydroxymuconic semialdehyde (5-CHMS) with λmax 380. The detection of catabolic genes involved in 4-CP degradation by PCR showed the presence of both mPH and catechol 2,3-dioxygenase (C23DO). Nucleotide sequence analysis of the alpha subunit of mPH from strain PT3 revealed specific phylogenetic grouping to known mPH. The metal coordination encoding regions from strain PT3 were found to be conserved with those from the homologous dinuclear oxo-iron bacterial monooxygenases. Two DE(D)XRH motifs was detected in LmPH of strain PT3 within an approximate 100 amino acid interval, a typical arrangement characteristic of most known PHs.
authors.affiliation: Biology Department, Faculty of Science, Taibah University, 344, Almadinah Almunwarrah, KSA, waelsme@yahoo.com.
authors.forename: Wael S
authors.initials: WS
authors.lastname: El-Sayed
authors.forename: Mohamed K
authors.initials: MK
authors.lastname: Ibrahim
authors.forename: Salama A
authors.initials: SA
authors.lastname: Ouf
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002733:Chlorophenols / Q000378:metabolism*; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D053858:Metabolic Networks and Pathways / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013011:Sorbic Acid / Q000031:analogs & derivatives; D013053:Spectrophotometry
chemical_list: D002733:Chlorophenols; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D021122:Protein Subunits; D012336:RNA, Ribosomal, 16S; C029107:4-chlorophenol; C020909:2-hydroxy-5-chloromuconic acid semialdehyde; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase; D013011:Sorbic Acid