Photinus pyralis Luciferase
Purified and characterized in 1978, Photinus pyralis luciferase (E.C. 1.13.12.7) is an enzyme found within the peroxisomes of the lantern organ located in the abdomen of the North American firefly (Photinus pyralis).[1] It is a member of an ANL superfamily which is made of acyl-CoA synthetases, non-ribosmal peptide synthetases (NRPSs), and luciferases. These enzymes all produce an acyl-AMP intermediate as part of their catalytic reactions.[2] Luciferases, along with a substrate luciferin, produce light by a reaction with ATP. Organisms that can do this include bacteria, fungi, algae, fish, squid, shrimp, and insects including the firefly.[3] Some uses of bioluminescence in nature: luring prey, mating and courtship or helping to camouflage by erasing the shadow and making it invisible from below.[4] In research labs, the reporter firefly luciferase from Photinus pyralis is widely used in molecular biology and small molecule high-throughput screening (HTS) assays.[5] Light production produced by this enzyme is a very sensitive analytical tool in detection and quantification of ATP, phosphate activity detection, as well as DNA sequencing. It also has applications in public health, specifically in detection of microorganisms. The use of luciferase in monitoring gene expressions, tumor growth, and metastasis has been studied more recently.[6]
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 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 antiparallel β-barrel (green) as well as two β-sheet subdomains (pink and blue) that create a five-layered αβαβα tertiary structure.[1] The C-terminal domain, on the other hand, is folded into an α+β tertiary structure (yellow).[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][7] This enclosement creates a hydrophobic environment which prevents light production from being quenched by water.[1][8]
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.[9] 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.[10] The luciferin binding pocket is comprised of the residues 341GLT343, 346TSA348, 245HHGFGMT251 (helix), 315GGA317 (loop), and R218.[10] 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.[10] 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.[10]
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 CO2 and 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.[11]
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. [5][11]
File:Luciferase Mechanism Without Spelling Errors.jpg
The active site environment influences the wavelength of the light emitted. Single amino acid changes within the active site of Photinus pyralis luciferase can shift the luminescence from yellow-green to red. 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.[4]
Function
There are three main functions of bioluminescence in nature: offense, defense and communication. Offense suggests baiting or enticing prey, defense suggests camouflage or protection and communication relates to courtship and mating. The literature suggests that the firefly species mainly use bioluminescence for communication purposes.<ref name=Greer2002> Greer, L.F., and Szalay, A.A. (2002). “Imaging of Light Emission from the Expression of Luciferases in Living Cells and Organisms: A Reivew.” Luminescence 17(1):43-74. doi: 10.1002/bio.676. Cite error: The opening <ref> tag is malformed or has a bad name== 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 1.4 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
- ↑ Sundlov J.A., Fontaine D.M., Southworth T.L., Branchini B.R., and Gulick, A.M. (2012) “Crystal structure of firefly luciferase in a second catalytic conformation supports a domain alternation mechanism”, Biochemistry 51(33): 6493-6495. doi: 10.1021/bi300934s
- ↑ Amani-Bayat Z., Hosseinkhani S., Jafari R., and Khajeh K. (2012) “Relationship between stability and flexibility in the most flexible region Photinus pyralis luciferase”, Biochim. Biophy. Acta 1842(2): 350-358. doi 10.1016/j.bbapap.2011.11.003
- ↑ 4.0 4.1 Shapiro E., Lu C., and Baneyx F. (2005) “A Set of Multicolored Photinus Pyralis Luciferase Mutants for in Vivo Bioluminescence Applications”, PEDS 18(12): 581-587. doi:10.1093/protein/gzi066.
- ↑ 5.0 5.1 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
- ↑ Riahi-Madvar, A. and Hosseinkhani, S. (2009) “Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis”, PEDS 22(11):655-663. doi:10.1093/protein/gzp047.
- ↑ 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
- ↑ 10.0 10.1 10.2 10.3 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
- ↑ 11.0 11.1 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.
