Functional characterization and molecular modeling of methylcatechol 2,3-dioxygenase from o-xylene-degrading Rhodococcus sp. strain DK17.
Rhodococcus sp. strain DK17 is known to metabolize o-xylene and toluene through the intermediates 3,4-dimethylcatechol and 3- and 4-methylcatechol, respectively, which are further cleaved by a common catechol 2,3-dioxygenase. A putative gene encoding this enzyme (akbC) was amplified by PCR, cloned, and expressed in Escherichia coli. Assessment of the enzyme activity expressed in E. coli combined with sequence analysis of a mutant gene demonstrated that the akbC gene encodes the bona fide catechol 2,3-dioxygenase (AkbC) for metabolism of o-xylene and alkylbenzenes such as toluene and ethylbenzene. Analysis of the deduced amino acid sequence indicates that AkbC consists of a new catechol 2,3-dioxygenase class specific for methyl-substituted catechols. A computer-aided molecular modeling studies suggest that amino acid residues (particularly Phe177) in the beta10-beta11 loop play an essential role in characterizing the substrate specificity of AkbC.
Kim
Dockyu
D
Department of Biology, Institute of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
Chae
Jong-Chan
JC
Jang
Jung Yeon
JY
Zylstra
Gerben J
GJ
Kim
Young Min
YM
Kang
Beom Sik
BS
Kim
Eungbin
E
D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D050557:Catechol 2,3-Dioxygenase; D049308:Dioxygenases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012240:Rhodococcus / Q000145:classification / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014992:Xylenes / Q000378:metabolism*
D011994:Recombinant Proteins; D014992:Xylenes; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase; C026114:2-xylene