Sandbox GGC3: Difference between revisions
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==Firefly Luciferase== | ==Firefly Luciferase== | ||
<StructureSection loadfiles='4G36''4G37' size='340' side='right' caption='Luciferin-4-monooxygenase. The wild-type luciferase in the adenylate-forming conformation with DLSA (PDB 4G36) and the cross-linked luciferase in the second catalytic conformation with DLSA (PDB 4G37)' scene=''> | <StructureSection loadfiles='4G36''4G37' size='340' side='right' caption='Luciferin-4-monooxygenase. The wild-type luciferase in the adenylate-forming conformation with DLSA (PDB 4G36) and the cross-linked luciferase in the second catalytic conformation with DLSA (PDB 4G37)' scene=''> | ||
Firefly luciferase, of the common eastern firefly (''Photinus pyralis''), is responsible for the ability of the firefly to exhibit bioluminescence. The enzyme luciferin-4-monoxygenase, which catalyzes a multistep oxidative decarboxylation of the luciferyl-AMP intermediate (LH<sub>2</sub>-AMP) to produce bioluminescence, is a part of the ANL superfamily named so after the '''a'''cyl-CoA syntheses, the adenylation domains of the modular '''n'''on-ribosomal peptide synthetases (NRPs), and '''l'''uciferase. | Firefly luciferase, of the common eastern firefly (''Photinus pyralis''), is responsible for the ability of the firefly to exhibit bioluminescence. The enzyme luciferin-4-monoxygenase, which catalyzes a multistep oxidative decarboxylation of the luciferyl-AMP intermediate (LH<sub>2</sub>-AMP) to produce bioluminescence, is a part of the ANL superfamily named so after the '''a'''cyl-CoA syntheses, the adenylation domains of the modular '''n'''on-ribosomal peptide synthetases (NRPs), and '''l'''uciferase. | ||
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The ANL enzymes catalyze two-step reactions: the first an adenylating step in which an acyl-AMP intermediate is produced; the second step in which the adenylate then serves as a substrate for the multistep oxidative decarboxylation of the luciferyl-AMP (LH<sub>2</sub>-AMP) intermediate, resulting in bioluminescence. | The ANL enzymes catalyze two-step reactions: the first an adenylating step in which an acyl-AMP intermediate is produced; the second step in which the adenylate then serves as a substrate for the multistep oxidative decarboxylation of the luciferyl-AMP (LH<sub>2</sub>-AMP) intermediate, resulting in bioluminescence. | ||
ANL enzymes follow a domain alternation strategy for the first adenylation reaction, in which the reaction is catalyzed by <scene name='75/752266/4g36/2'>one conformation</scene>, and following the formation of the adenylate intermediate and release of pyrophosphate (PPi), the C-terminal domain undergoes a rotational transformation that is necessary for <scene name='75/752266/4g37/2'>the second partial reaction</scene>. The <scene name='75/752266/Active_site/1'>active site</scene><ref name=“Branchini”>Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., Helgerson, L. C., & Zimmer, M. (1999). Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color. ''Biochemistry 38''(40), 13223–13230. https://doi.org/10.1021/bi991181o</ref> of ANL enzymes resides between a 400-500 residue N-terminal domain and a smaller C-terminal domain of ~110-130 amino acids<ref name="Sundlov">Sundlov, J. A., Fontaine, D. M., Southworth, T. L., Branchini, B. R., Gulick, A. M. (2012). Crystal Structure of Firefly Luciferase in a Second Catalytic Conformation Supports a Domain Alternation Mechanism. ''Biochemistry 51''(33), 6493-6495. https://doi.org/10.1021/bi300934s</ref>. Ten conserved regions of these proteins have been termed the A1-A10 motifs which play critical roles in either or both partial reactions<ref name="Marahiel">Marahiel, M. A., Stachelhaus, T., Mootz, H. D. (1997). Modular Peptide Synthetases Involved in Nonribosmal Peptide Synthesis. ''Chemical Reviews 97''(7), 2651-2674. https://doi.org/10.1021/cr960029e</ref>. Two lysine residues are required for each partial reaction, suggestive that luciferase similarly adopts a rotational transformation for complete catalysis. A mutation of <scene name='75/752266/Lys529/1'>Lys529</scene>, the A10 lysine, impairs only the initial adenylation reaction<ref name="Sundlov"/> whereas mutation of <scene name='75/752266/Lys443/1'>Lys443</scene> in the A8 region disrupts the oxidative reaction<ref name="Sundlov"/>. | ANL enzymes follow a domain alternation strategy for the first adenylation reaction, in which the reaction is catalyzed by <scene name='75/752266/4g36/2'>one conformation</scene>, and following the formation of the adenylate intermediate and release of pyrophosphate (PPi), the C-terminal domain undergoes a rotational transformation that is necessary for <scene name='75/752266/4g37/2'>the second partial reaction</scene>. The <scene name='75/752266/Active_site/1'>active site</scene><ref name=“Branchini”>Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., Helgerson, L. C., & Zimmer, M. (1999). Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color. ''Biochemistry 38''(40), 13223–13230. https://doi.org/10.1021/bi991181o</ref> of ANL enzymes resides between a 400-500 residue N-terminal domain and a smaller C-terminal domain of ~110-130 amino acids<ref name="Sundlov">Sundlov, J. A., Fontaine, D. M., Southworth, T. L., Branchini, B. R., & Gulick, A. M. (2012). Crystal Structure of Firefly Luciferase in a Second Catalytic Conformation Supports a Domain Alternation Mechanism. ''Biochemistry 51''(33), 6493-6495. https://doi.org/10.1021/bi300934s</ref>. Ten conserved regions of these proteins have been termed the A1-A10 motifs which play critical roles in either or both partial reactions<ref name="Marahiel">Marahiel, M. A., Stachelhaus, T., & Mootz, H. D. (1997). Modular Peptide Synthetases Involved in Nonribosmal Peptide Synthesis. ''Chemical Reviews 97''(7), 2651-2674. https://doi.org/10.1021/cr960029e</ref>. Two lysine residues are required for each partial reaction, suggestive that luciferase similarly adopts a rotational transformation for complete catalysis. A mutation of <scene name='75/752266/Lys529/1'>Lys529</scene>, the A10 lysine, impairs only the initial adenylation reaction<ref name="Sundlov"/> whereas mutation of <scene name='75/752266/Lys443/1'>Lys443</scene> in the A8 region disrupts the oxidative reaction<ref name="Sundlov"/>. | ||
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[[Image:Mechanism_of_Firefly_Bioluminescence.png|thumb|upright=2.3|The generally accepted mechanism of firefly bioluminescence. The first reaction (1) involves the production of an luciferyl-adenylate intermediate. The second reaction (2) involves oxidative decarboxylation that emits CO<sub>2</sub> and results in bioluminescent properties<ref name="Sundlov"/>.]] | [[Image:Mechanism_of_Firefly_Bioluminescence.png|thumb|upright=2.3|The generally accepted mechanism of firefly bioluminescence. The first reaction (1) involves the production of an luciferyl-adenylate intermediate. The second reaction (2) involves oxidative decarboxylation that emits CO<sub>2</sub> and results in bioluminescent properties<ref name="Sundlov"/>.]] | ||
The first partial reaction entails the conversion of the carboxyl group of <small>D</small>-luciferin<ref name="Sundlov"/><ref name="Bruce">Branchini, B. R., Southworth, T. L., Murtiahsaw, M. H., Wilkinson, S. R., Khattak, N. F., Rosenberg, J. C., & Zimmer, M. (2005). Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain. “Biochemistry 44”(5), 1385-1393. https://doi.org/10.1021/bi047903f</ref><ref name="Nakamura">Nakamura, M., Maki, S., Amano, Y., Ohkita, Y., Niwa, K., Hirano, T., Ohmiya, Y., & Niwa, H. (2005). Firefly luciferase exhibits bimodal action depending on the luciferin chirality. “Biochemical and Biophysical Research Communications, 331”(2), 471–475. https://doi.org/10.1016/j.bbrc.2005.03.202</ref> by luciferase in the presence of ATP and Mg<sup>2+</sup>, yielding luciferyl-adenylate (LH<sub>2</sub>-AMP) and pyrophosphate as a by-product. Amino acid residues subsequently are recruited to promote the oxidation of LH<sub>2</sub>-AMP using molecular oxygen by luciferase (acting as a monooxygenase)<ref name="Oba">Oba, Y., Ojika, M., Inouye, S. (2003). Firefly luciferase is a bifunctional enzyme: ATP-dependent monoxygenase and a long chain fatty acyl-CoA synthetase. “FEBS Letters 540”(1-3), 251-254. https://doi.org/10.1016/S0014-5793(03)00272-2</ref>, which then eventually yields oxyluciferin in the excited-state and CO<sub>2</sub>. It is upon the return from the excited-state to the ground state that the emittance of a yellow-green light is observed (λ≈560 nm)<ref name=Nakamura/>. | The first partial reaction entails the conversion of the carboxyl group of <small>D</small>-luciferin<ref name="Sundlov"/><ref name="Bruce">Branchini, B. R., Southworth, T. L., Murtiahsaw, M. H., Wilkinson, S. R., Khattak, N. F., Rosenberg, J. C., & Zimmer, M. (2005). Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain. “Biochemistry 44”(5), 1385-1393. https://doi.org/10.1021/bi047903f</ref><ref name="Nakamura">Nakamura, M., Maki, S., Amano, Y., Ohkita, Y., Niwa, K., Hirano, T., Ohmiya, Y., & Niwa, H. (2005). Firefly luciferase exhibits bimodal action depending on the luciferin chirality. “Biochemical and Biophysical Research Communications, 331”(2), 471–475. https://doi.org/10.1016/j.bbrc.2005.03.202</ref> by luciferase in the presence of ATP and Mg<sup>2+</sup>, yielding luciferyl-adenylate (LH<sub>2</sub>-AMP) and pyrophosphate as a by-product. Amino acid residues subsequently are recruited to promote the oxidation of LH<sub>2</sub>-AMP using molecular oxygen by luciferase (acting as a monooxygenase)<ref name="Oba">Oba, Y., Ojika, M., & Inouye, S. (2003). Firefly luciferase is a bifunctional enzyme: ATP-dependent monoxygenase and a long chain fatty acyl-CoA synthetase. “FEBS Letters 540”(1-3), 251-254. https://doi.org/10.1016/S0014-5793(03)00272-2</ref>, which then eventually yields oxyluciferin in the excited-state and CO<sub>2</sub>. It is upon the return from the excited-state to the ground state that the emittance of a yellow-green light is observed (λ≈560 nm)<ref name="Nakamura"/>. | ||
An alternative mechanism involving the enantiomer of <small>D</small>-luciferin exists, though typically <small>L</small>-luciferin acts as a competitive inhibitor to the bioluminescence-producing reaction<ref name=“Seliger”>Seliger, H. H., McElroy, W. D., White, E. H., & Field, G. F. (1961). Stereospecificity and firefly bioluminescence, a comparison of natural and synthetic luciferins. ‘’Proceedings of the National Academy of Sciences of the United States of America 47’’(8), 1129-1134. https://doi.org/10.1073/pnas.47.8.1129</ref>, though accounts of light production in small quantities have previously been reported<ref name=“Lembert”>Lembert, N. (1996). Firefly luciferase can use L-luciferin to produce light. ‘’Biochemical Journal 317’’(1), 273-277. https://doi.org/10.1042/bj3170273</ref>. The mechanism by which <small>L</small>-luciferin acts as the substrate in the presence of luciferase (and ATP and Mg<sup>2+</sup>) is the same in the first partial reaction, with both producing the intermediate luciferyl-adenylate. Rather than the oxidative decarboxylation step, the adenyl group (AMP) is substituted with CoA-SH yielding luciferyl-CoA. Furthermore, the stereospecificity of luciferase has shown that even in the presence of CoA-SH, <small>D</small>-luciferin was not converted into luciferyl-CoA but proceeded in being used for the emittance of light<ref name="Nakamura"/>. | |||