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{{Sandbox_gvsu_chm463}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --> | <!--{{Sandbox_gvsu_chm463}}--><!-- PLEASE ADD YOUR CONTENT BELOW HERE --> | ||
=''Photinus pyralis'' Luciferase= | =''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'').<ref name=Conti1996>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</ref> It is a member of an '''ANL''' superfamily which is made of <big>'''a'''</big>cyl-CoA synthetases, <big>'''n'''</big>on-ribosmal peptide synthetases (NRPSs), and <big>'''l'''</big>uciferases. These enzymes all produce an acyl-AMP intermediate as part of their catalytic reactions.<ref name=Sundlov2012>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</ref> 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.<ref name=Amani2012>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</ref> Some uses of bioluminescence in nature are luring prey, mating and courtship, or helping to camouflage the organism by erasing its shadow or making it invisible from below.<ref name=Shapiro2005>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.</ref> ''Photinus pyralis'' luciferase is used in a variety of analytic biological tests as well. | 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'').<ref name=Conti1996>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</ref> It is a member of an '''ANL''' superfamily which is made of <big>'''a'''</big>cyl-CoA synthetases, <big>'''n'''</big>on-ribosmal peptide synthetases (NRPSs), and <big>'''l'''</big>uciferases. These enzymes all produce an acyl-AMP intermediate as part of their catalytic reactions.<ref name=Sundlov2012>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</ref> 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.<ref name=Amani2012>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</ref> Some uses of bioluminescence in nature are luring prey, mating and courtship, or helping to camouflage the organism by erasing its shadow or making it invisible from below.<ref name=Shapiro2005>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.</ref> ''Photinus pyralis'' luciferase is used in a variety of analytic biological tests as well. | ||
<StructureSection load='1lci' size=' | <StructureSection load='1lci' size='350' side='right' background='none' scene='69/691535/Overall_structure_rainbow/4' caption='Structure of ''Photinus pyralis'' luciferase (PDB code [[1lci]])'> | ||
== Structure == | == Structure == | ||
''Photinus pyralis'' luciferase is a monomeric enzyme composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two <scene name='69/691535/Colored_domains/2'>domains</scene> (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.<ref name=Conti1996 /> The C-terminal domain, on the other hand, is folded into an α+β tertiary structure (yellow).<ref name=Conti1996 /> 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.<ref name=Conti1996 /><ref name=Marques2009>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</ref> This enclosement creates a hydrophobic environment which prevents light production from being quenched by water.<ref name=Conti1996 /><ref name=Bedford2012>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</ref> | ''Photinus pyralis'' luciferase is a monomeric enzyme composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two <scene name='69/691535/Colored_domains/2'>domains</scene> (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.<ref name=Conti1996 /> The C-terminal domain, on the other hand, is folded into an α+β tertiary structure (yellow).<ref name=Conti1996 /> 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.<ref name=Conti1996 /><ref name=Marques2009>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</ref> This enclosement creates a hydrophobic environment which prevents light production from being quenched by water.<ref name=Conti1996 /><ref name=Bedford2012>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</ref> | ||
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.<ref name=Branchini1998>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</ref><ref name=Zako2003>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</ref> 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.<ref name=Branchini1998 /> The luciferin binding pocket is comprised of the residues 341GLT343, 346TSA348, 245HHGFGMT251 (helix), 315GGA317 (loop), and R218.<ref name=Branchini1998 /> A model of the active site with a bound | 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.<ref name=Branchini1998>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</ref><ref name=Zako2003>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</ref> 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.<ref name=Branchini1998 /> The luciferin binding pocket is comprised of the residues 341GLT343, 346TSA348, 245HHGFGMT251 (helix), 315GGA317 (loop), and R218.<ref name=Branchini1998 /> A model of the active site with a bound molecule of tetraethylene glycol is shown <scene name='69/691535/Active_site_structure/2'>here</scene> (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.<ref name=Branchini1998 /> 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.<ref name=Branchini1998 /> | ||
</StructureSection> | </StructureSection> | ||
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In the ''Photinus pyralis'' luciferase reaction 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 O<sub>2</sub> 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 CO<sub>2</sub> collection from the reaction was obtainable. It was stated that the CO<sub>2</sub> produced was pumped directly out of the reaction. This was not possible due to the high reaction rate of CO<sub>2</sub> and tert-butoxide ion and the stability of monoalkyl carbonates. Johnson and Shimomura determined that an oxygen atom that makes up the CO<sub>2</sub> does indeed stem from the O<sub>2</sub> 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.<ref name=White1980>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.</ref> | In the ''Photinus pyralis'' luciferase reaction 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 O<sub>2</sub> 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 CO<sub>2</sub> collection from the reaction was obtainable. It was stated that the CO<sub>2</sub> produced was pumped directly out of the reaction. This was not possible due to the high reaction rate of CO<sub>2</sub> and tert-butoxide ion and the stability of monoalkyl carbonates. Johnson and Shimomura determined that an oxygen atom that makes up the CO<sub>2</sub> does indeed stem from the O<sub>2</sub> 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.<ref name=White1980>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.</ref> | ||
Below is a schematic representation of the ''Photinus pyralis'' lucerifase reaction.<ref name=Branchini1998 /> In the first step, ''Photinus pyralis'' luciferase catalyzes the production of luciferyl-AMP from luciferin and ATP. An inorganic pyrophosphate is released in this step. In the second step, ''Photinus pyralis'' luciferase converts luciferyl-AMP and O<sub>2</sub> into oxyluciferin* and CO<sub>2</sub>.<ref name=White1980 /><ref name=Thorne2012>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:http://dx.doi.org/10.1016%2Fj.chembiol.2012.07.015</ref> When the excited electrons in oxyluciferin* relax, light is emitted. | Below is a proposed schematic representation of the ''Photinus pyralis'' lucerifase reaction.<ref name=Branchini1998 /> In the first step, ''Photinus pyralis'' luciferase catalyzes the production of luciferyl-AMP from luciferin and ATP. An inorganic pyrophosphate is released in this step. As of 2010, the biosynthesis of luciferin was still undetermined; however, cysteine appears to be one of the reactants.<ref name=Inouye2010>Inouye, S. (2010) "Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions", Cell. Mol. Life Sci. 67(3): 387-404. doi: 10.1007/s00018-009-0170-8</ref> In the second step, ''Photinus pyralis'' luciferase converts luciferyl-AMP and O<sub>2</sub> into oxyluciferin* and CO<sub>2</sub>.<ref name=White1980 /><ref name=Thorne2012>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:http://dx.doi.org/10.1016%2Fj.chembiol.2012.07.015</ref> When the excited electrons in oxyluciferin* relax, light is emitted. | ||
[[Image:Luciferin_Mechanism.jpg]] | [[Image:Luciferin_Mechanism.jpg]] | ||