1. Article
* E-mail: ssliuhl@263.net
Received March 27, 2013; published May 2, 2013.
Supporting information for this article is available free of charge via the Internet at http://sioc-journal.cn.
Project supported by the National Natural Science Foundation of China (Nos. 21177097, 20977065) and Specialized Research Fund for the Doctoral Program
of Higher Education (20120072110052).
(Nos. 21177097, 20977065) (No. 20120072110052) .
Acta Chim. Sinica 2013, 71, 1035—1040 © 2013 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences http://sioc-journal.cn 1035
*
( 200092)
.
. (Vibrio qinghaiensis sp.-Q67, Q67)
, . ,
, Q67 .
Q67 . , Q67 , /
, / . /
. Q67 ,
.
; ; ; ;
Chen, Fu Liu, Shushen* Duan, Xintian
(Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and
Engineering, Tongji University, Shanghai 200092)
Abstract Bioluminescence technique derived from the luciferase-based catalization reactions has been widely used in
chemistry, biological assay and environmental science. The three-dimensional crystal structures of luciferase proteins in some
firefly and luminescent bacteria were elucidated. Vibrio qinghaiensis sp.-Q67 (Q67), one of freshwater luminescent bacteria
which was extracted from Gymnocypris przewalskii in Qinghai lake, has been used in biological assay and toxicity evaluation
of many chemicals. However, the crystal structure of the luciferase (the most important catalyzer to bioluminescent) in Q67 is
still not established, which hinders the process of the study on the molecular mechanism of toxicities of chemicals to Q67. In
this study, the three dimensional structure of bacterial luciferase in Q67 was constructed by using the heterodimeric homol-
ogy modeling combined with the molecular dynamics simulation which were performed with explicit TIP3P water. The
simulation system was equilibrated at 4 ns, and was prolonged for another 4 ns for extracting the equilibrium trajectories at
the 8th ns. The stability of the system was monitored through the convergences of energy, temperature, and global root mean
square deviation (RMSD). The ptraj modules in the AMBER software were used to analyze hydrogen bond occupancy be-
tween and subunit. And then, the molecular mechanics generalized born surface area method was applied to identify
critical amino acids of the and subunits that interact with each other during the native heterotetrameric structure forma-
tion. It was shown that the luciferase in Q67 is a heterdimer including two polypeptide subunits ( and ) and the stabilization
of this heterodimer was mainly determined by the van der waals force. The specificity of association is realized by hydrogen
bonds formed between subunits. However, the electrostatic interaction from the net charge on and subunit is unfavorable
to the stability of the dimer. The active sites of flavin mononucleotide binding to the luciferase in Q67 are located in the ac-
tive pocket of subunit. The subunit is helpful to keep the structural stability of the active sites on the subunit.
Keywords heterodimer; luciferase; homology modeling; freshwater luminescent bacteria; molecular dynamics
1
[1]
. (luciferin)
(aliphatic aldehyde) .
(luciferase assay) .
,
(bioluminescence).

2. 1036 http://sioc-journal.cn © 2013 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences Acta Chim. Sinica 2013, 71, 1035—1040
,
DNA . ,
[2 4]
.
. ,
N ( 4 436) C ( 440 544),
[5]
. Mg2
ATP O2 , (Luciferin) ,
.
, luxA
luxB [6]
.
(FMNH2) ,
. ,
[7,8]
.
,
. Auld [2,9]
Photinus pyralis
,
, . Sundlov [10]
Photinus
pyralis ,
C 140 N ,
(Luciola cruciate) [11]
. Campbell [8]
Vibrio harveyi
[12]
loop (262
291) , -FMN
, ,
. Yamasaki [13]
Vibrio fischeri C12H25OH
C11H23CHO , C12H25OH
.
,
(Vibrio qinghaiensis sp.-Q67, Q67)[14]
pH (6.5 11) ,
[15]
. Q67
[16 18]
.
Q67 [19 22]
.
Q67
Hormesis [23]
.
.
, Q67
,
. , Q67 ,
[24]
Q67
,
, (FMN)
, .
2
2.1
Q67 8 ns
. 1 , 4 ns , ,
4 8 ns (RMSD) , RMSD
2.27 Å, 0.3 Å,
.
, , 4 ns
.
1 Q67 RMSD
Figure 1 Plot of root mean square deviation (RMSD) vs. time for the
luciferase in Q67
2.2 Q67 Ramachandran
8 ns Q67
, Ramachandran ( ), 2 .
2 Q67
Figure 2 Ramachandran plot of the structure of luciferase in Q67
, Lys283 ,
Ile219 , Asp147 Ile230 ,
. Gly
Gly ,

3. Acta Chim. Sinica 2013, 71, 1035—1040 © 2013 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences http://sioc-journal.cn 1037
phi psi .
N-C ,
, - ,
. Q67 ,
, ( )
532 ( 87.4%) ( )73 (12%)
( )3 (0.5%) 1 (0.2%).
Q67 .
2.3 Q67
Q67 .
3 - 15 - 22 - 1 -
. 2 - 13 - 24 -
. 3 Q67 .
2.3.1
3 3a . 1
A 9 , 2 B
2 , 3 C 2
. A 6 ,
1 2 Rossman .
C 2 (Lys268 Tyr270
Trp277 Asp279 ). 50 (
14.1%), 144 ( 40.6%).
2.3.2
2 3b . ,
1 D , 9 , 2
E 2 . D
5 , 1 2
Rossman . E (Tyr143
Cys144 Ile156 Ser157 ). 39
( 12.0%), 143 ( 44.1%).
, ,
32%.
, ,
,
. .
2.3.3
(RMSF) ,
( 4). 3 ,
Ser145 Gln159 ,
loop , Leu213
Ile219 , 3 Asp279
Arg291 loop
. ;
,
.
RMSF ( 4),
Met1 Asp195 RMSF ,
3 Q67 (a) (b)
Figure 3 Topological graphs for the secondary structures of (a) and
subunit (b) of the luciferase in Q67
, ,
, .
Arg201 Gln324 RMSF , .
Trp194 Asn198 ,
, ,
, RMSF .
2.4 /
Q67 5.
S1.
8 ns ,
, 90%
1, S1. 17 8
ns 90% 90%, 17

4. 1038 http://sioc-journal.cn © 2013 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences Acta Chim. Sinica 2013, 71, 1035—1040
4 Q67 RMSF
Figure 4 Plot of the root mean square fluctuation (RMSF) vs. the resi-
due index
5 Q67 /
Figure 5 The tertiary structure and binding pockets of the luciferase in
Q67
. Arg85 Glu43 , Glu43
Arg85 His45 Glu88 99%
, (
20.06, 17.72 21.73 kcal/mol). 3
( 1).
(∆G)
(∆Gpol-sol) (∆Gnpol-sol)
(∆Evdw) (∆Eele). ,
1
Table 1 The occupancy of hydrogen bonds formed between and
subunits and bonding distance
/% /Å
Asn159 @O Gln17 @N 100 2.90
Gln95 @O Thr18 @O 100 2.68
Glu43 @O Arg85 @NH2 100 2.79
Glu43 @O Arg85 @NH1 100 2.81
Gln95 @O Thr18 @O 100 2.74
Val57 @O Ser60 @O 100 2.71
Glu43 @O Arg85 @N 100 2.75
Ser152 @O Arg115 @N 99 2.93
Glu88 @O His45 @N 99 2.76
Ser152 @O Gln266 @N 97 2.94
Asn273 @O Asn118 @N 97 2.93
Thr80 @O Arg85 @N 97 3.01
Gln266 @O Asn145 @N 96 3.03
Thr80 @O Arg85 @N 96 2.89
Glu48 @O Asn159 @N 95 2.91
His152 @O Asn118 @N 94 3.02
Phe116 @O Val82 @N 91 2.96
water@O Thr80 @O 97 2.90
water@O Asn273 @N 97 3.01
water@O Ser298 @O 93 2.79
.
[25]
. 4
(His45 -Glu88 , Arg85 -Glu43 , Glu43 -Arg85
Lys98 -Asp22 ) ( 97%),
( 10
kcal/mol). ( 2) ,
( 1) ,
. ,
, , 3
Arg85 -His76 , Arg85 -His45 His45 -Arg85 ( 2).
183.0 kcal/mol,
∆Gnpol-sol 39.6 kcal/mol) ∆Evdw 309.3 kcal/mol)
∆Gpol-sol 45.0 kcal/mol , .
∆Eele 210.9 kcal/mol, . /
22 [7,26]
. ,
,
, 165.9 kcal/mol,
Q67 .
,
, 348.9 kcal/mol. , /
,
. / /

5. Acta Chim. Sinica 2013, 71, 1035—1040 © 2013 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences http://sioc-journal.cn 1039
2 a
Table 2 The interaction energies of pairwise residues between and
subunits
1 2 ∆Gvdw ∆Gele ∆Gpol sol ∆Gnpol sol ∆Gtot
His45 Glu88 0.61 53.61 31.85 0.64 21.79
Arg85 Glu43 1.20 49.14 28.32 0.49 20.12
Glu43 Arg85 0.81 44.70 26.48 0.32 17.73
Lys98 Asp22 0.55 50.93 40.44 0.79 10.73
Thr80 Arg85 0.21 11.60 2.94 0.69 9.57
Asp89 Thr47 0.49 11.52 4.60 0.68 7.11
Lys98 Thr18 0.47 7.86 2.90 1.24 6.66
Glu88 His45 0.64 39.97 34.71 0.65 6.55
Arg85 His81 1.74 3.97 0.65 1.38 6.44
Glu48 Asn159 0.55 8.35 3.47 0.88 6.31
Arg85 His76 0.56 4.37 1.41 0.40 2.00
Arg85 His45 0.34 30.19 25.74 0.29 3.82
His45 Arg85 0.61 33.67 26.62 0.55 5.89
a
: kcal/mol.
,
.
2.5 Q67
Q67 , 2
A B ( 5). A 852.75 Å3
,
, B 635.75 Å3
,
.
. Q67 FMN ,
FMN Vibrio harveyi
. FMN Vibrio
harveyi ,
Glu43, Ala75, Arg107, Leu109, Ser176
Thr179. Q67 Vibrio harveyi
(84%), ,
, , Vibrio harveyi
FMN ,
Q67 FMN . Q67
6.
FMN ( ) Glu43 ,
Ala75 , Arg107 , Leu109 , Glu175 Ser176 ,
FMN . Thr179
, ,
FMN . FMN , Thr179
FMN 2.47 Å,
. Ser145 Arg165 loop ,
Thr179 .
, . RMSF ,
. ,
.
6 Q67 ( ) FMN ( )
Figure 6 The structures of active pockets of the binding-free luciferase
(blue) and the binding FMN-luciferase (yellow) in Q67
3
Q67
/ ,
.
, ,
. FMN
Vibrio harveyi ,
Q67 FMN . ,
,
. Q67
.
4
4.1
Q67 , 355
, 324 ,
[27,28]
.
Q67 PDB ,
30% 3fgc[8]
, 1bsl[6]
,
1nfp[29]
1fvp[30]
, Vibrio harveyi
3fgc Q67 ( 84%,
60%). 3fgc HOMCOS (http://
strcomp.protein.osaka-u.ac.jp/homcos/)
. S2.
(Zseqcon 53.6) .
UCSF Chimera[31]
MODELLER[32]
Q67
loop .
4.2
AMBER Q67
[33]
. ff12SB ,
antechamber tool .
TIP3P ,

6. 1040 http://sioc-journal.cn © 2013 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences Acta Chim. Sinica 2013, 71, 1035—1040
8.5 Å. 22 Na .
, ,
(isothermal-isobaric ensemble, NPT)
[34]
. : ,
, .
2000 1000
[35]
. 50 ps 0 K 300 K,
NPT . PME (particle
meshewald) .
8 Å. Langevin thermostat
300 K. SHAKE , 2
fs[36]
. 10 ps . ptraj
Q67 RMSD
RMSF .
8 ns Q67
. 8 ns 8 ns
, . S3.
4.3 MM/GBSA
MM/GBSA , 8 ns
100 ,
[25]
. ∆G
[37]
. ,
GB (Generalized Born)
;
SASA LCPO [38]
.
,
AMBER MMPBSA.py
MM/GBSA , .
References
[1] Razgulin, A.; Ma, N.; Rao, J. H. Chem. Soc. Rev. 2011, 40, 4186.
[2] Thorne, N.; Shen, M.; Lea, W. A.; Simeonov, A.; Lovell, S.; Auld, D.
S.; Inglese, J. Chem. Biol. 2012, 19, 1060.
[3] Loening, A. M.; Fenn, T. D.; Gambhir, S. S. J. Mol. Biol. 2007, 374,
1017.
[4] Aufhammer, S. W.; Warkentin, E.; Ermler, U.; Hagemeier, C. H.;
Thauer, R. K.; Shima, S. Protein Sci. 2005, 14, 1840.
[5] Conti, E.; Franks, N. P.; Brick, P. Structure 1996, 4, 287.
[6] Thoden, J. B.; Holden, H. M.; Fisher, A. J.; Sinclair, J. F.;
Wesenberg, G.; Baldwin, T. O.; Rayment, I. Protein Sci. 1997, 6, 13.
[7] Ke, D. C.; Tu, S. C. Photochem. Photobiol. 2011, 87, 1346.
[8] Campbell, Z. T.; Weichsel, A.; Montfort, W. R.; Baldwin, T. O.
Biochemistry 2009, 48, 6085.
[9] Auld, D. S.; Lovell, S.; Thorne, N.; Lea, W. A.; Maloney, D. J.;
Shen, M.; Rai, G.; Battaile, K. P.; Thomas, C. J.; Simeonov, A.;
Hanzlik, R. P.; Inglese, J. Proc. Natl. Acad. Sci. U. S. A. 2010, 107,
4878.
[10] Sundlov, J. A.; Fontaine, D. M.; Southworth, T. L.; Branchini, B. R.;
Gulick, A. M. Biochemistry 2012, 51, 6493.
[11] Nakatsu, T.; Ichiyama, S.; Hiratake, J.; Saldanha, A.; Kobashi, N.;
Sakata, K.; Kato, H. Nature 2006, 440, 372.
[12] Fisher, A. J.; Thompson, T. B.; Thoden, J. B.; Baldwin, T. O.;
Rayment, I. J. Biol. Chem. 1996, 271, 21956.
[13] Yamasaki, S.; Yamada, S.; Takehara, K. Anal. Sci. 2013, 29, 41.
[14] Zhu, W. J.; Zheng, T. L.; Li, W. M. Luminous Bacteria and
Environmental Monitoring, China Light Industry Press, Beijing,
2009. ( , , , ,
, , 2009.)
[15] Bureau of Environmental Protection People's Republic of China,
Monitoring And Analysis Methods about Water And Wastewater, 4th
ed., China Environmental Science Press, Beijing, 2002. (
, ( ), ,
, 2002.)
[16] Li, J.; Jiang, G. X.; Yang, B.; Dong, X. H.; Feng, L. Y.; Lin, S.;
Chen, F.; Ashraf, M.; Jiang, Y. M. Anal. Bioanal. Chem. 2012, 402,
1347.
[17] Ma, X. Y.; Wang, X. C.; Liu, Y. J. J. Hazard. Mater. 2011, 190, 100.
[18] Liu, S. S.; Zhang, J.; Zhang, Y. H.; Qin, L. T. Acta Chim. Sinica
2012, 70, 1511. ( , , , , , 2012,
70, 1511.)
[19] Liu, S. S.; Song, X. Q.; Liu, H. L.; Zhang, Y. H.; Zhang, J.
Chemosphere 2009, 75, 381.
[20] Dou, R. N.; Liu, S. S.; Mo, L. Y.; Liu, H. L.; Deng, F. C. Environ.
Sci. Pollut. Res. 2011, 18, 734.
[21] Zhu, X. W.; Liu, S. S.; Ge, H. L.; Liu, Y. Water Res. 2009, 43, 1731.
[22] Zhang, J.; Liu, S.-S.; Yu, Z.-Y.; Zhang, J. Chemosphere 2013, 91,
462.
[23] Wang, L. J.; Liu, S. S.; Yuan, J.; Liu, H. L. Chemosphere 2011, 84,
1440.
[24] Cai, W.; Christophe, C. Acta Chim. Sinica 2013, 71, 159. ( ,
Christophe Chipot, , 2013, 71, 159.)
[25] Baris, I.; Tuncel, A.; Ozber, N.; Keskin, O.; Kavakli, I. H. PLoS
Comput. Biol. 2009, 5, 1.
[26] Taveecharoenkool, T.; Angsuthanasombat, C.; Kanchanawarin, C.
PMC Biophys. 2010, 3, 10.
[27] Fukuhara, N.; Go, N.; Kawabata, T. Biophysics 2007, 3, 13.
[28] Fukuhara, N.; Kawabata, T. Nucleic Acids Res. 2008, 36, W185.
[29] Moore, S. A.; James, M. N. G. J. Mol. Biol. 1995, 249, 195.
[30] Kita, A.; Kasai, S.; Miyata, M.; Miki, K. Acta Crystallogr. Sect. D:
Biol. Crystallogr. 1996, 52, 77.
[31] Yang, Z.; Lasker, K.; Schneidman-Duhovny, D.; Webb, B.; Huang,
C. C.; Pettersen, E. F.; Goddard, T. D.; Meng, E. C.; Sali, A.; Ferrin,
T. E. J. Struct. Biol. 2012, 179, 269.
[32] Fiser, A.; Do, R. K. G.; Sali, A. Protein Sci. 2000, 9, 1753.
[33] Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.;
Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J.
J. Comput. Chem. 2005, 26, 1668.
[34] Han, M.; Zhang, J. Z. H. J. Chem. Inf. Model. 2010, 50, 136.
[35] Khuntawee, W.; Rungrotmongkol, T.; Hannongbua, S. J. Chem. Inf.
Model. 2012, 52, 76.
[36] Chen, X.; Zhao, X.; Wang, S.; Wang, L.; Li, W.; Sun, C. Acta Chim.
Sinica 2013, 71, 199. ( , , , , ,
, , 2013, 71, 199.)
[37] Hou, T. J.; Wang, J. M.; Li, Y. Y.; Wang, W. J. Chem. Inf. Model.
2011, 51, 69.
[38] Greenidge, P. A.; Kramer, C.; Mozziconacci, J. C.; Wolf, R. M. J.
Chem. Inf. Model. 2013, 53, 201.
(Cheng, B.; Fan, Y.)