title: HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866.
abstract: In addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg(-1) and showed apparent Km values of 11.40 ± 3.05 μM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 μM with NADH and similar Km values for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 μM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China.
authors.forename: Hong-Jun
authors.initials: HJ
authors.lastname: Chao
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China.
authors.forename: Yan-Fei
authors.initials: YF
authors.lastname: Chen
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China.
authors.forename: Ti
authors.initials: T
authors.lastname: Fang
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Engineering Science, University of Oxford, Oxford, United Kingdom.
authors.forename: Wei E
authors.initials: WE
authors.lastname: Huang
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China n.zhou@pentium.whiov.ac.cn.
authors.forename: Ning-Yi
authors.initials: NY
authors.lastname: Zhou
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010795:Phthalic Acids / Q000378:metabolism*; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014992:Xylenes / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D062385:Hydroxybenzoates; D010795:Phthalic Acids; D014992:Xylenes; C009091:protocatechuic acid; C027804:2,4-dimethylphenol; D006899:Mixed Function Oxygenases; C021399:4-hydroxyisophthalate hydroxylase; C042941:4-hydroxyisophthalic acid