Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24.
Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of approximately 340 and approximately 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the approximately 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the approximately 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The Km of TID for indene was 380 microM. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The Km of TID for naphthalene was 28 microM. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.
Priefert
Horst
H
Department of Biology, 68-370, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
O'Brien
Xian M
XM
Lessard
Philip A
PA
Dexter
Annette F
AF
Choi
Ellen E
EE
Tomic
Sladjana
S
Nagpal
Geeta
G
Cho
Jennie J
JJ
Agosto
Melina
M
Yang
Lucy
L
Treadway
Sheri L
SL
Tamashiro
Lance
L
Wallace
Matthew
M
Sinskey
Anthony J
AJ
D002851:Chromatography, High Pressure Liquid; D004790:Enzyme Induction; D007192:Indenes / Q000378:metabolism*; D009876:Operon / Q000235:genetics; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D007192:Indenes; D010105:Oxygenases; C023172:toluene dioxygenase