1
86
D.-H. Kim et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 179–187
the Korea Science and Engineering Foundation [Grant R01-2008-
00-21072-02008]; and the Second Stage BK21 Project from the
0
Ministry of Education, Science and Technology of the Republic of
Korea.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2010.01.017.
References
[
[
[
1] F.P. Guengerich, Chem. Res. Toxicol. 22 (2009) 237–238.
2] V.B. Urlacher, S. Eiben, Trends Biotechnol. 24 (2006) 324–330.
3] C.H. Yun, K.H. Kim, D.H. Kim, H.C. Jung, J.G. Pan, Trends Biotechnol. 25 (2007)
289–298.
[
[
[
[
4] B.M. van Vugt-Lussenburg, E. Stjernschantz, J. Lastdrager, C. Oostenbrink, N.P.
Vermeulen, J.N. Commandeur, J. Med. Chem. 50 (2007) 455–461.
5] M.D. Johnson, H. Zuo, K.H. Lee, J.P. Trebley, J.M. Rae, R.V. Weatherman, Z. Desta,
D.A. Flockhart, T.C. Skaar, Breast Cancer Res. Treat. 85 (2004) 151–159.
6] C.R. Otey, G. Bandara, J. Lalonde, K. Takahashi, F.H. Arnold, Biotechnol. Bioeng.
9
3 (2006) 494–499.
7] D.H. Kim, K.H. Kim, K.H. Liu, H.C. Jung, J.G. Pan, C.H. Yun, Drug Metab. Dispos.
6 (2008) 2166–2170.
Fig. 7. Superimposition of the structures of CYP102A1 (yellow) and mutant #15
violet) complexes with their substrates (phenacetin and 7-ethoxyresorufin). The
superimposition shows that the -sheet structures constituting the entry site for
the substrate change conformation in the mutant (circle). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of
the article.)
3
(
[8] D.H. Kim, T. Ahn, H.C. Jung, J.G. Pan, C.H. Yun, Drug Metab. Dispos. 37 (2009)
932–936.
[9] M. Landwehr, L. Hochrein, C.R. Otey, A. Kasrayan, J.E. Backvall, F.H. Arnold, J.
Am. Chem. Soc. 128 (2006) 6058–6059.
[10] A.M. Sawayama, M.M. Chen, P. Kulanthaivel, M.S. Kuo, H. Hemmerle, F.H.
Arnold, Chemistry 15 (2009) 11723–11729.
[
[
[
11] M.C. Damsten, J.S. de Vlieger, W.M. Niessen, H. Irth, N.P. Vermeulen, J.N. Com-
mandeur, Chem. Res. Toxicol. 21 (2008) 2181–2187.
12] M.C. Damsten, B.M. van Vugt-Lussenburg, T. Zeldenthuis, J.S. de Vlieger, J.N.
Commandeur, N.P. Vermeulen, Chem. Biol. Interact. 171 (2008) 96–107.
13] E. Stjernschantz, B.M. van Vugt-Lussenburg, A. Bonifacio, S.B. de Beer, G. van der
Zwan, C. Gooijer, J.N. Commandeur, N.P. Vermeulen, C. Oostenbrink, Proteins
conformation by the amino acid substitutions (Fig. 7). Further, side
chain movements are required to optimally fit phenacetin and 7-
ethoxyresorufin into the active sites of CYP102A1 and mutant #15.
These side chain adjustments resulting from the decreased cavity
size may mechanistically be related to the origin of the cavity size
of active sites.
7
1 (2008) 336–352.
14] B.M. Lussenburg, L.C. Babel, N.P. Vermeulen, J.N. Commandeur, Anal. Biochem.
41 (2005) 148–155.
[
3
[
[
15] F.P. Guengerich, T. Shimada, Mutat. Res. 400 (1998) 201–213.
16] S. Sansen, J.K. Yano, R.L. Reynald, G.A. Schoch, K.J. Griffin, C.D. Stout, E.F. Johnson,
J. Biol. Chem. 282 (2007) 14348–14355.
4
. Conclusion
[
17] D.J. Waxman, T.K. Chang, Methods Mol. Biol. 320 (2006) 153–156.
This study examined the interaction of a set of CYP102A1
[18] C.H. Yun, G.P. Miller, F.P. Guengerich, Biochemistry 39 (2000) 11319–11329.
[
19] A.B. Carmichael, L.L. Wong, Eur. J. Biochem. 268 (2001) 3117–3125.
mutants and some typical substrates of human CYP1A2 and
revealed that bacterial CYP102A1 enzymes catalyze the same
reactions as human CYP1A2. The oxidations of phenacetin, 7-
methoxyresorufin, and 7-ethoxyresorufin (typical substrates of
human CYP1A2) were catalyzed by some mutants of CYP102A1.
In the case of PhOD reactions, one major product, acetaminophen,
was produced as a result of the human CYP1A2 reaction. The other
biological hydroxylated product, acetol, was not produced with
the bacterial enzyme. Acetaminophen formation was confirmed
by HPLC analysis and LC–MS, comparing the metabolite with the
authentic acetaminophen compound. Catalytic activities of some
CYP102A1 mutants were higher than those of human CYP1A2.
Also, some mutants of CYP102A1 were not inhibited by ␣NF, a
known competitive inhibitor of human CYP1A2. The similar oxi-
dation profiles and highly catalytic activities of CYP102A1 mutants
on human CYP1A2 substrates suggest that these bacterial enzymes
may represent a model system for studying the human enzymes.
The computational findings further imply that a conformational
change in the active site cavity size is related to the change in activ-
ity in the CYP102A1 mutants. It can be proposed that the activity
change results from movement of several residues in the active
site.
[
20] Q.S. Li, J. Ogawa, R.D. Schmid, S. Shimizu, Appl. Environ. Microbiol. 67 (2001)
5735–5739.
[21] T. Omura, R. Sato, J. Biol. Chem. 239 (1964) 2379–2385.
[
[
22] M.D. Burke, R.T. Mayer, Chem. Biol. Interact. 45 (1983) 243–258.
23] J. Liu, S.S. Ericksen, M. Sivaneri, D. Besspiata, C.W. Fisher, G.D. Szklarz, Arch.
Biochem. Biophys. 424 (2004) 33–43.
[24] D.H. Kim, K.H. Kim, E.M. Isin, F.P. Guengerich, H.Z. Chae, T. Ahn, C.H. Yun, Protein
Expr. Purif. 57 (2008) 188–200.
[
[
25] E.M. Isin, F.P. Guengerich, J. Biol. Chem. 281 (2006) 9127–9136.
26] G. Vriend, J. Mol. Graph. 8 (1990) 52–56, 29.
[27] A. Sali, T.L. Blundell, J. Mol. Biol. 234 (1993) 779–815.
[
28] Y. Duan, C. Wu, S. Chowdhury, M.C. Lee, G. Xiong, W. Zhang, R. Yang, P. Cieplak,
R. Luo, T. Lee, J. Caldwell, J. Wang, P. Kollman, J. Comput. Chem. 24 (2003)
1999–2012.
[29] J. Wang, R.M. Wolf, J.W. Caldwell, P.A. Kollman, D.A. Case, J. Comput. Chem. 25
(2004) 1157–1174.
[
[
30] D.L. Harris, J.Y. Park, L. Gruenke, L. Waskell, Proteins 55 (2004) 895–914.
31] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheese-
man, J.A. Montgomery, Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S.
Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa,
M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P.
Hratchian, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Strat-
mann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K.
Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich,
A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari,
J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Ste-
fanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith,
M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. John-
son, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, revision C.02,
Inc., Wallingford, CT, 2004.
Acknowledgments
[
32] P. Rydberg, L. Olsen, P.O. Norrby, U. Ryde, J. Chem. Theory Comput. 3 (2007)
1
765–1773.
This work was supported in part by the 21C Frontier Micro-
bial Genomics and the Application Center Program of the Ministry
of Education, Science and Technology of the Republic of Korea;
[
[
33] L. Tian, R.A. Friesner, J. Chem. Theory Comput. 5 (2009) 1421–1431.
34] D.A. Pearlman, D.A. Case, J.W. Caldwell, W.S. Ross, T.E. Cheatham, S. Debolt, D.
Ferguson, G. Seibel, P. Kollman, Comput. Phys. Commun. 91 (1995) 1–41.