Functional characterization of ent-kaurene oxidase, MtKO, from Montanoa tomentosa (Zoapatle)
Kaurene oxidases are P450 proteins that catalyze the conversion of ent-kaurene into kaurenoic acid, the final enzymatic product with a wide range of pharmacological properties. We describe the functional characterization of an ent-kaurene oxidase (EC 1.14.13.78) isolated from Montanoa to...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
University of Belgrade, University of Novi Sad
2015-01-01
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| Series: | Archives of Biological Sciences |
| Subjects: | |
| Online Access: | http://www.doiserbia.nb.rs/img/doi/0354-4664/2015/0354-46641400024V.pdf |
| Summary: | Kaurene oxidases are P450 proteins that catalyze the conversion of
ent-kaurene into kaurenoic acid, the final enzymatic product with a wide
range of pharmacological properties. We describe the functional
characterization of an ent-kaurene oxidase (EC 1.14.13.78) isolated from
Montanoa tomentosa after heterologous expression in Saccharomyces
cerevisiae, as well as the detection of the enzymatic activity in the plant
itself. In the presence of NADPH and FAD, the microsomal fraction from
transformed INVSc1 cells, ent-kaurene produced ent-kaurenoic acid, which was
confirmed by GC-MS analyses. The kinetic parameters for ent-kaurene using
0.5 mg of microsomal protein were Km app= 80.63±1.2 μM and V max app=
31.80±1.8 μmol-1mg-1h-1. Optimal temperature and pH were 30°C and 7.6,
respectively. Similar kinetic parameters were observed when leaf microsomes
from M. tomentosa were assayed under the same conditions as for yeast
microsomes. This result strongly suggests that ent-kaurene oxidase activity
is present in leaf microsomes. The enzymatic activity was competitively
inhibited by paclobutrazol, with IC50=43.9 μM, implying that MtKO is
resistant to inhibition by azolic-type compounds. This study confirmed the
biochemical detection of ent-kaurene oxidase activity in the plant, and the
heterologous functionality of a cDNA with an ent-kaurene oxidase identity
from M. tomentosa (zoapatle). |
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| ISSN: | 0354-4664 1821-4339 |