Benzylic and aryl hydroxylations of m-xylene by o-xylene dioxygenase from Rhodococcus sp. strain DK17

• Published in: Applied microbiology and biotechnology Vol. 86; no. 6; pp. 1841 - 1847
• Main Authors: Kim, Dockyu, Choi, Ki Young, Yoo, Miyoun, Choi, Jung Nam, Lee, Choong Hwan, Zylstra, Gerben J, Kang, Beom Sik, Kim, Eungbin
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.05.2010; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Amino Acid Sequence; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Benzyl Alcohols - metabolism; Biological and medical sciences; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Catalysis; Cloning; Dioxygenases - chemistry; Dioxygenases - genetics; Dioxygenases - metabolism; E coli; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gas chromatography; Gas Chromatography-Mass Spectrometry; Genetics; Hydroxylation; Life Sciences; Mass spectrometry; Metabolites; Microbial Genetics and Genomics; Microbiology; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Oxidation-Reduction; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Rhodococcus - enzymology; Rhodococcus - genetics; Studies; Thermal cycling; Xylene; Xylenes - chemistry; Xylenes - metabolism; Xylene dioxygenase; Benzylic hydroxylation; effect; Hydroxylation; Xylene; Bacteria; Rhodococcus; Meta effect; Actinomycetes; o-Xylene dioxygenase
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-009-2418-5

Escherichia coli cells expressing Rhodococcus DK17 o-xylene dioxygenase genes were used for bioconversion of m-xylene. Gas chromatography-mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol in the ratio 9:1. Molecular modeling suggests that o-xylene dioxygenase can hold xylene isomers at a kink region between α6 and α7 helices of the active site and α9 helix covers the substrates. m-Xylene is unlikely to locate at the active site with a methyl group facing the kink region because this configuration would not fit within the substrate-binding pocket. The m-xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3-methylbenzylalcohol could be formed, presumably due to the meta effect. Alternatively, the m-xylene molecule can rotate counterclockwise, allowing the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol. Site-directed mutagenesis combined with structural and functional analyses suggests that the alanine-218 and the aspartic acid-262 in the α7 and the α9 helices play an important role in positioning m-xylene, respectively.