Benzylic and aryl hydroxylations of m-xylene by o-xylene dioxygenase from Rhodococcus sp. strain DK17.
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 alpha6 and alpha7 helices of the active site and alpha9 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 alpha7 and the alpha9 helices play an important role in positioning m-xylene, respectively.
Kim
Dockyu
D
Polar BioCenter, Korea Polar Research Institute, KORDI, Incheon, 406-840, South Korea.
Choi
Ki Young
KY
Yoo
Miyoun
M
Choi
Jung Nam
JN
Lee
Choong Hwan
CH
Zylstra
Gerben J
GJ
Kang
Beom Sik
BS
Kim
Eungbin
E
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001592:Benzyl Alcohols / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D006900:Hydroxylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D014992:Xylenes / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D001592:Benzyl Alcohols; D011994:Recombinant Proteins; D014992:Xylenes; C027804:2,4-dimethylphenol; D049308:Dioxygenases; C551337:o-xylene dioxygenase, Rhodococcus; C031285:3-xylene; C042289:3-methylbenzyl alcohol; C026114:2-xylene