Construction of chimeric catechol 2,3-dioxygenase exhibiting improved activity against the suicide inhibitor 4-methylcatechol.
Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI). When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-toluate because 4-methylcatechol, a metabolite of p-toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on p-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-toluate.
Okuta
Akiko
A
Marine Biotechnology Institute, Heita, Kamaishi, Iwate 026-0001, Japan.
Ohnishi
Kouhei
K
Harayama
Shigeaki
S
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D050557:Catechol 2,3-Dioxygenase; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D049308:Dioxygenases; D007700:Kinetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002396:Catechols; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C018599:4-methylcatechol; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase