Reaction of Lentinula edodes P450 Produced in Yeast
83
By using the heterologous protein expression system
of S. cerevisiae, the substrates of the basidiomycete L.
edodes P450 enzyme (Le.CYP1) (produced in the yeast)
were found to be 7-ethoxycoumarin and benzo(a)pyrene.
Although the cytochrome P450-mediated biotransfor-
mation of benzo(a)pyrene has been reported in the
basidiomycete Phanerochaete chrysosporium and Pleu-
rotus pulmonarius,16,19) the catalytic action on both of
benzo(a)pyrene and 7-ethoxycoumarin is first case as far
as we know. The system we developed in this paper
should be helpful to determine the endogenous sub-
strates of L. edodes P450 enzyme, and to clarify the
physiological function in L. edodes of the P450 enzyme.
Acknowledgments
This work was partly supported by a research grant
from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan.
References
1) Akiyama, R., Sato, Y., Kajiwara, S., and Shishido, K.,
Cloning and expression of cytochrome P450 genes,
belonging to a new P450 family, of the basidiomycete
Lentinula edodes. Biosci. Biotechnol. Biochem., 66,
2183–2188 (2002).
2) Gonzalez, F. J., and Korzekwa, K. R., Cytochromes
P450 expression systems. Annu. Rev. Pharmacol. Tox-
icol., 35, 369–390 (1995).
3) Oeda, K., Sakaki, T., and Ohkawa, H., Expression of rat
liver cytochrome P-450MC cDNA in Saccharomyces
cerevisiae. DNA, 4, 203–210 (1985).
4) Kalb, V. F., Loper, J. C., Dey, C. R., Woods, C. W., and
Sutter, T. R., Isolation of a cytochrome P-450 structural
gene from Saccharomyces cerevisiae. Gene, 45, 237–
245 (1986).
Fig. 3. HPLC Analysis of Conversion of 7-Ethoxycoumarin into 7-
Hydroxycoumarin by the Le.CYP1-Containing Microsomal Fraction
of INVSc1[pYES2-HA-Le.cyp1].
5) Skaggs, B. A., Alexander, J. F., Pierson, C. A.,
Schweitzer, K. S., Chun, K. T., Koegel, C., Barbuch,
R., and Bard, M., Cloning and characterization of the
Saccharomyces cerevisiae C-22 sterol desaturase gene,
encoding a second cytochrome P-450 involved in
ergosterol biosynthesis. Gene, 169, 105–109 (1996).
6) Briza, P., Eckerstorfer, M., and Breitenbach, M., The
sporulation-specific enzymes encoded by the DIT1 and
DIT2 genes catalyze a two-step reaction leading to a
soluble LL-dityrosine-containing precursor of the yeast
spore wall. Proc. Natl. Acad. Sci. USA, 91, 4524–4528
(1994).
7) Yabusaki, Y., Artificial P450/reductase fusion enzymes:
what can we learn from their structures? Biochimie, 77,
594–603 (1995).
8) Kolodziej, P. A., and Young, R. A., Epitope tagging and
protein surveillance. Methods Enzymol., 194, 508–519
(1991).
The 7-ethoxycoumarin was added to the Le.CYP1-containing
microsomal fraction of INVSc1[pYES2-HA-Le.cyp1]. The mixtures
were extracted with ethyl acetate before (panel C) and after 60-min
incubation at 30ꢀC (panel D), and the resulting extracts were
analyzed. The 7-ethoxycoumarin alone (panel A) and 7-hydrox-
ycoumarin alone (panel B) were similarly analyzed. For further
details, see Materials and Methods.
the decrease of the amount of benzo(a)pyrene, but the
microsomal fraction containing no Le.CYP1 did not
(data not shown). The rate of decrease of benzo(a)pyr-
ene was to be 13 pmoles minꢂ1ꢁg of microsomal
proteinꢂ1 (Table 2). The reaction product, however,
could not be measured for benzo(a)pyrene. The decrease
of the amount of benzo(a)pyrene was inhibited by
carbon monoxide, which can bind to the haem and
exclude substrate. So the reaction was clear to be
catalyzed by Le.CYP1. There is the possibility that some
reaction product(s) which is highly reactive to a
biomolecule and is not recovered by extraction with
ethyl acetate was generated.
9) Ito, H., Jukuda, Y., Murata, K., and Kimura, A.,
Transformation of intact yeast cells treated with alkali
cations. J. Bacteriol., 153, 163–168 (1983).
10) Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,
Seidman, J. G., Smith, J. A., and Struhl, K., ‘‘Current
Protocols in Molecular Biology’’, Greene Publishing