*For correspondence. E-mail: waelsme@yahoo.com; Tel.: +8460008-1433 Wael S. El-Sayed 1,2 * , Mohamed K. Ibrahim 2 , and Salama A. Ouf 1,3 1 Biology Department, Faculty of Science, Taibah University, 344, Almadinah Almunwarrah, KSA 2 Microbiology Department, Faculty of Science, Ain Shams University, 11566, Cairo, Egypt 3 Botany Department, Faculty of Science, Cairo University, Giza 12613, Egypt (Received May 6, 2013 / Accepted August 13, 2013) Journal of Microbiology (2014) Vol. 52, No. 1, pp. Copyright ⓒ 2013, The Microbiological Society of Korea DOI 10.1007/s12275-014-3250-x Molecular Characterization of the Alpha Subunit of Multicomponent Phenol Hydroxylase from 4-Chlorophenol-Degrading Pseudomonas sp. Strain PT3 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 hy- droxylation to the corresponding chlorocatechol. This hy- pothesis was supported by polymerase chain reaction (PCR) detection of the LmPH encoding sequence and UV/VIS spec- trophotometric analysis of the culture filtrate showing ac- cumulation of 5-chloro-2-hydroxymuconic semialdehyde (5-CHMS) with λ max 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 homolo- gous 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. Keywords : 4-chlorophenol, biodegradation, phenol hydroxy- lase, catalytic domain, Pseudomonas Introduction Since their discovery, chloroaromatics represented a serious source of potentially hazard materials for both humans and the environment. Tens of thousands of such chemicals have been developed and have invariably found their way into the environment. Some are not readily biodegradable and become persistent. Because of their various health effects and the subsequent increase in public concern, careful moni- toring and regulation of these pollutants is now generally required. Chlorinated phenols are common environmental contaminants; they have been extensively used as biocides, detergents, wood preservatives, bleaching agents, and disin- fectants. They are subsequently released into the environment as by-products from many industrial activities (Tartakovsky et al., 2001). Chemical, physical, and biological methods have been es- tablished for remediation purposes; however, an integrated chemical-biological and/or physical system for treatment of phenolic wastes could be very useful. In this regard, advanced oxidation processes like wet air oxidation, catalytic wet air oxidation, H2O2-promoted, catalytic wet air oxidation, ac- tivated carbon were found to be a useful option to prepare the phenolic effluents before the biological remediation (Esplugas et al., 2002; Rubalcaba et al., 2007). Biological degradation of chlorinated phenols has been re- ported and pathways for their degradation have also been elucidated (Harayama and Rekik, 1989; Arenghi et al., 2001). The key step in the degradation of a phenolic compound was found to be the initial hydroxylation via phenol hy- droxylase (PH). PHs are known to initiate the catabolism of a vast array of phenolic compounds by hydroxylation to the corresponding catechols (Evans et al., 1971; Schwien and Schmidt, 1982). Such partially oxidized aromatic inter- mediates may undergo ortho or meta cleavage via catechol 1,2-dioxygenase (C12DO) or C23DO, respectively. The pro- duced metabolites would then be mineralized to carbon dio- xide and water in the subsequent reactions of the tricar- boxylic acid cycle (Powlowski and Shingler, 1994; Cafaro et al., 2004). Enzymatic removal of phenolic compounds has been inves- tigated. It has been shown that peroxidases are able to react with aqueous phenolic compounds and form non-soluble materials that could be easily removed from the aqueous phase, however; these processes suffer from enzyme inacti- vation (Bodalo et al., 2007; Ulson de Souza et al., 2007; Zhang et al., 2007). Bacterial multicomponent monooxygenases are a diverse