SELECTIVE HYDROXYLATION OF NAPHTHALENE USING THE H O -DEPENDENT ENGINEERED P450BM3 DRIVEN
5
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the current study. DFSMs play a critical role in generating
peroxygenase activity of P450BM3 for naphthalene
hydroxylation as we expected. However, to our surprise,
the substitution of Thr268 with Val268 in the F87A
mutant of P450BM3 resulted in 3fold improvement in
the catalytic TON. This is quite different from the results
we observed in the epoxidation of styrene by the DFSM
facilitated P450BM3H O system [14]. In that case, the
Acknowledgments
This work was supported by NSFC (No. 21778060),
Shandong Provincial Key Laboratory of Synthetic
Biology (No. SPKLSB201702), and the Qingdao
Innovative Leading Talent Project (to Z. Cong).
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Supporting information
TON of F87A/T268V was almost 4 times lower than
that of F87A in the presence of ImC6Phe. That result
is easier to understand because Thr268 is well known to
play a role in the formation of active species compound
I in the catalytic cycle of P450BM3 [5, 29–30]. We
have also indirectly confirmed this possible function of
Thr268 due to the complete loss of peroxygenase activity
of the F87A/T268V mutant of P450BM3 in the absence
of DFSMs. The reason why the hydroxylation activity of
naphthalene is improved by the introduced Val268 is still
unclear. One possible explanation is that the combination
of mutations at positions 268 and 87 efficiently adjusts the
active site pocket size to accommodate the naphthalene
substrate. Indeed, the TON of F87G/T268V was further
improved 11fold over F87A as observed in this study.
It has ever been reported that the mutation of Ala74 and
Leu188 together with Phe87 dramatically improved the
oxidation of polycyclic aromatic hydrocarbons including
naphthalene in the NADPHdependent P450BM3 system
Figures S1 and S2 are given in the supplementary
material. This material is available free of charge via the
Internet at http://www.worldscinet.com/jpp/jpp.shtml.
REFERENCES
1. Bernhardt R and Urlacher VB. Appl. Microbiol.
Biotechnol. 2014; 98: 6185–6203.
2. Guengerich FP and Munro AW. J. Biol. Chem.
2013; 288: 17065–17073.
3. Fasan R. ACS Catal. 2012; 2: 647–666.
4. Ortiz de Montellano PR. Chem. Rev. 2010; 110:
932–948.
5. Whitehouse CJ, Bell SG and Wong LL. Chem. Soc.
Rev. 2012; 41: 1218–1260.
6. Liu Q,YangY, Li H, Zhu R, Shao Q,Yang S and Xu
J. Biosens. Bioelectron. 2015; 64: 147–153.
7. Holtmann D, Fraaije MW, Arends IW, Opperman
DJ and Hollmann F. Chem. Commun. 2014; 50:
13180–13200.
[
26–27]. Accordingly, we prepared the mutants A74G/
F87A and F87A/L188Q to examine the hydroxylation
activity of naphthene using the current H O system.
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2
However, comparable TONs were observed only for
these two mutants in comparison with F87A (Table 1).
These results partially supported the assertion that
the residue at position 268 played a unique role in the
hydroxylation activity of naphthalene by the DFSM
facilitated P450BM3H O system.
8. Shoji O, Fujishiro T, Nakajima H, Kim M, Nagano
S, Shiro Y and Watanabe Y. J. Biol. Inorg. Chem.
2014; 19: 529–539.
9. Munro AW, McLean KJ, Grant JL and Makris TM.
Biochem. Soc. Trans. 2018; 46: 183–196.
10. Cirino PC and Arnold FH. Angew. Chem. Int. Ed.
2
2
2
003; 115: 3299–3301.
1
1. Kumar S, Chen CS, Waxman DJ and Halpert JR.
CONCLUSION
J. Biol. Chem. 2005; 280: 19569–19575.
In summary, we have shown for the first time that
the engineered P450BM3 could catalyze the H O
12. Behera RK, Goyal S and Mazumdar S. J. Inorg.
Biochem. 2010; 104: 1185–1194.
2
2
dependent hydroxylation of naphthalene with the
assistance of DFSMs. This study increases the potential
of cytochrome P450s for developing the practical
hydroxylation biocatalysts using the DFSMfacilitated
P450H O system by the avoidance of their complicated
13. Onoda H, Shoji O and Watanabe Y. Dalton. Trans.
2015; 44: 15316–15323.
14. Ma N, Chen Z, Chen J, Chen J, Wang C, Zhou H,
Yao L, Shoji O, Watanabe Y and Cong Z. Angew.
Chem. Int. Ed. 2018; doi: 10.1002/ange.201801592,
in press.
2
2
electron transport chains. The mutation of the residue at
position 268 unexpectedly improved the H O dependent
15. Wang Y, Lan D, Durrani R and Hollmann F. Curr.
Opin. Chem. Biol. 2017; 37: 1–9.
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2
hydroxylation activity in the current system. The unique
role of Val268 may be further understood with the
help of Xray structure analysis, molecular dynamics
simulations, and theoretical calculations. Such work and
further studies aimed at improving the hydroxylation
activity of aromatic hydrocarbons including but not
limited to naphthalene are in progress by combining
additional mutations in the DFSMfacilitated P450H O
16. Olmedo A and Aranda C, Del Río JC, Kiebist J,
Scheibner K, Martínez AT and Gutiérrez A. Angew.
Chem. Int. Ed. 2016; 128: 12248–12251.
17. Rittle J and Green MT. Science 2010; 330: 933–937.
18. Omura T and Sato R. J. Biol. Chem. 1964; 239:
2370–2378.
19. Iwasaki M, Juvonen R, Lindberg R and Negeshi M.
J. Biol. Chem. 1991; 266: 3380–3392.
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2
system.
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