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Photinus Pyralis Luciferase
Structure
Photinus pyralis luciferase a monomeric enzyme composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two domains (the N-terminal domain and the C-terminal domain) by a wide cleft. Although not shown in the model, the domains (N-terminal in green and C-terminal in blue) are connected by a flexible loop structure. The N-terminal domain (residues 4-436) is much larger than the C-terminal domain (residues 440-544) and is formed by an antiparallet β-barrel, two β-sheets, and two α-helices.[1] The secondary structures and motif are arranged to form a five-layered, alternating αβ tertiary structure. The C-terminal domain, on the other hand, is folded into an α+β tertiary structure.[1] Currently, it is thought that the active site is located at the surfaces where the domains meet and that a conformation change occurs after the substrates are bound in which the domains come together and enclose the substrates.[1][2] This enclosement creates a hydrophobic environment which prevents light production from being quenched by water.[1][3]
A model for the active site of Photinus pyralis luciferase was proposed by Branchini and colleagues in 1998 and has held up to more recent data.[4] In this model, the enzyme contains a binding pocket for ATP as well as a binding pocket for luciferin. The binding pocket for ATP is formed by the residues 316GAP318, 339GYGL342, and V362, and binds to the adenine ring.[5] The luciferin binding pocket is comprised of the residues 341GLT343, 346TSA348, 245HHGFGMT251 (helix), 315GGA317 (loop), and R218.[5] A model of the active site with a bound luciferase inhibitor (PTC128) is shown here (blue=ATP binding pocket, purple=luciferin binding pocket, and green=residues shared by binding pockets). The S314-L319 loop and Q338-A348 region were found to be in different positions when substrates were bound.[5] Since the loop blocks both of the binding pockets when in the unbound state, it makes sense that a conformational change in the loop must occur.[5]
Mechanism
It was believed that the chemically produced excited states stemmed from dioxetanone. This idea was proposed based on a common type of chemiluminescence which required O2 at certain points in which dioxetanone is a precursor to the excited state. De Luca and colleagues did a study that proposed that the dioxetanone mechanism for bio- and chemiluminescence were false. Their experiment used oxygen isotopes and concluded that the oxygen atoms that the produced carbon dioxide consisted of did not stem from the consumed oxygen. This study, however, has been analyzed and several flaws have been discovered such as, incomplete chain of events and no proof of CO2 collection from the reaction was obtainable. It was stated that the CO2 produced was pumped directly out of the reaction. This was not possible due to the high reaction rate of CO2and tert-butoxide ion and the stability of monoalkyl carbonates. Johnson and Shimomura determined that an oxygen atom that makes up the CO2 does indeed stem from the O2 consumed by the reaction in firefly bioluminescence. De Luca and colleagues reevaluated their work and their results agreed with Johnson and Shimomura. Therefore, the dioxetane-dioxetanone mechanism for firefly bioluminescence and chemiluminescence is supported.[6]
Step one: In the photinus pyralis, the reaction begins with luciferin. Luciferase catalyzes ATP and magnesium ion to produce luciferyl AMP from luciferin.
Step 2: Luciferase then catalyzes O2, producing light and oxyluciferin from Luciferyl AMP. [7][6]
[[Image: Luciferase_Mechanism_Without_Spelling_Errors.jpg][8]
The active site environment influences the wavelength of the light emitted. Single amino acid changes within the active site of Photinus pyralis can shift the luminescence from yellow-green to red.[8] Modifying the position of the Ser314-Leu319 loop near the active site can alter Biolumanescence color. When assayed under acidic conditions, all spectra underwent a red shift while basic conditions caused a blue shift. These experiments were done using E. Coli as the host organism indicating that the internal pH of the cell was close to the external pH. These findings suggest a possible use of bioluminescence in pH monitoring, biosensing and tissue and animal imaging. (Shapiro, 2005)
Function
Lab Use
This is a sample scene created with SAT to color by Group, and another to make a transparent representation of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.
- ↑ 1.0 1.1 1.2 1.3 Conti E., Franks N.P., Brick P. (1996) "Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes", Structure 4(3): 287-298. doi: 10.1016/S0969-2126(96)00033-0
- ↑ Marques S.M. and Esteves da Silva J.C.G. (2009) "Firefly bioluminescence: mechanistic approach of luciferase catalyzed reactions", IUBMB Life 61(1): 6-17. doi: 10.1002/iub.134
- ↑ Bedford R., LePage D., Hoffman R., Kennedy S., Gutschenritter T., Bull L., Sujijantarat N., DiCesare J.C., and Sheaff R.J. (2012) "Luciferase inhibition by a novel naphthoquinone", J. Photochem. Photobiol., B 107: 55-64. doi: 10.1016/j.jphotobiol.2011.11.008
- ↑ Zako T., Ayabe K., Aburatani T., Kamiya N., Kitayama A., Ueda H., and Nagamune T. (2003) "Luminescent and substrate binding activities of firefly luciferase N-terminal domain", 1649(2): 183-189. doi: 10.1016/S1570-9639(03)00179-1
- ↑ 5.0 5.1 5.2 5.3 Branchini B.R., Magyar R.A., Murtiashaw M.H., Anderson S.M., and Zimmer M. (1998) "Site-directed mutagenesis of Histidine 245 in firefly luciferase: a proposed model of the active site", Biochemistry 37(44): 15311-15319. doi: 10.1021/bi981150d
- ↑ 6.0 6.1 White, E. H., Steinmetz, M. G., Miano, J. D., Wildes, P. D. and Morland, R. (1980) "Chemi- and bioluminescence of firefly luciferin", J. Am. Chem. Soc. 102(9): 3199-3208.
- ↑ Thorne, N., Shen, M., Lea, W. A., Simeonov, A., Lovell, S., Auld, D. S. and Inglese, J. (2012) "Firefly luciferase in chemical biology: A compendium of inhibitor, mechanistic evaluation of chemotypes, and suggested use as a reporter", Chem. Biol. 19(8): 1060-1072. doi:https://dx.doi.org/10.1016%2Fj.chembiol.2012.07.015
- ↑ 8.0 8.1 Auld, D.S., Southhall, N. T., Jadhav, A., Johnson, R. L., Diller, D. J., Simeonov, A., Austin, C. P., and Inglese, J. (2008) "Characteristics of chemical libraries for luciferase inhibitory activity", J. Med. Chem. 51(8):2372-2386. doi:10.1021/jm701302v
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