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==Firefly Luciferase==
==Firefly Luciferase==
<small>waluigi menacingly stares</small>
 
<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 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 L-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"/>.
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"/>.
 




Latest revision as of 20:06, 28 April 2021

Firefly LuciferaseFirefly Luciferase

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 (LH2-AMP) to produce bioluminescence, is a part of the ANL superfamily named so after the acyl-CoA syntheses, the adenylation domains of the modular non-ribosomal peptide synthetases (NRPs), and luciferase.

Function

The Common Eastern Firefly in a hand emitting a yellow hue, showing bioluminescence.[1]

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 (LH2-AMP) intermediate, resulting in bioluminescence.

ANL enzymes follow a domain alternation strategy for the first adenylation reaction, in which the reaction is catalyzed by , 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 . The [1] of ANL enzymes resides between a 400-500 residue N-terminal domain and a smaller C-terminal domain of ~110-130 amino acids[2]. Ten conserved regions of these proteins have been termed the A1-A10 motifs which play critical roles in either or both partial reactions[3]. Two lysine residues are required for each partial reaction, suggestive that luciferase similarly adopts a rotational transformation for complete catalysis. A mutation of , the A10 lysine, impairs only the initial adenylation reaction[2] whereas mutation of in the A8 region disrupts the oxidative reaction[2].


Biochemical Mechanism of LH2-AMP Oxidation

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 CO2 and results in bioluminescent properties[2].

The first partial reaction entails the conversion of the carboxyl group of D-luciferin[2][4][5] by luciferase in the presence of ATP and Mg2+, yielding luciferyl-adenylate (LH2-AMP) and pyrophosphate as a by-product. Amino acid residues subsequently are recruited to promote the oxidation of LH2-AMP using molecular oxygen by luciferase (acting as a monooxygenase)[6], which then eventually yields oxyluciferin in the excited-state and CO2. 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)[5].

An alternative mechanism involving the enantiomer of D-luciferin exists, though typically L-luciferin acts as a competitive inhibitor to the bioluminescence-producing reaction[7], though accounts of light production in small quantities have previously been reported[8]. The mechanism by which L-luciferin acts as the substrate in the presence of luciferase (and ATP and Mg2+) 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, D-luciferin was not converted into luciferyl-CoA but proceeded in being used for the emittance of light[5].


Structural highlights

5'-O-[N-(Dehydroluciferyl)-sulfamoyl] adenosine, shortened to DLSA for brevity. The sulfamate moiety is shown to the far left (sulfur atoms are represented in yellow while oxygen atoms are represented in red). Further, the carbonyl oxygen of the luciferyl-adenylate is connected to the sulfamate moiety via nitrogen atom (represented in blue). [2]
The φ(Phi)/ψ(Psi) angles of the Lys439 residue undergo a rotational transformation of −73°/−12° to −69°/158° in the adenylate-forming to second-catalytic conformation, respectively, showing that the major torsional change in the ψ angle is observed[2].

The conserved catalytic lysine for the adenylation reaction[9], Lys529, with the carbonyl oxygen of the adenylate, the O5 atom that bridges the ribose and sulfamate moiety, and the main chain carbonyl of Gly316. The second conformation observations show that the of Lys443 adopts a nearly identical position as Lys529, and Gln448 of the C-terminal domain rotates into the binding pocket where it with a sulfamate oxygen[2][4]. Altogether (with the inclusion of an between Glu479 and Arg437), these interactions are responsible for the stabilization of the new C-terminal conformation.


Relevance

The Common Eastern Firefly expressing bioluminescence seen giving off a yellow-green hue.[3]

Firefly luciferase has successfully been shown to act as modulatory bioluminescent indicator in the detection and quantification of protein kinase A activation in living cells [10]. Further, due to its bioluminescent sensitivity, firefly luciferase has been utilized in assays as a genetic reporter in eukaryotic cells[11][12][13], amongst other things.




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)

Drag the structure with the mouse to rotate

ReferencesReferences

  1. ↑ 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
  2. ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 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
  3. ↑ 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
  4. ↑ 4.0 4.1 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
  5. ↑ 5.0 5.1 5.2 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
  6. ↑ 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
  7. ↑ 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
  8. ↑ Lembert, N. (1996). Firefly luciferase can use L-luciferin to produce light. ‘’Biochemical Journal 317’’(1), 273-277. https://doi.org/10.1042/bj3170273
  9. ↑ Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Anderson, S. M. (2000). The Role of Lysine 529, a Conserved Residue of the Acyl-Adenylate-Forming Enzyme Superfamily, in Firefly Luciferase. Biochemistry 39(18), 5433-5440. https://doi.org/10.1021/bi9928804
  10. ↑ Sala-Newby, G. B., & Campbell, A. K. (1991). Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase. “The Biochemical Journal”, 279 (Pt 3), 727–732. https://doi.org/10.1042/bj2790727
  11. ↑ de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., & Subramani, S. (1987). Firefly luciferase gene: structure and expression in mammalian cells. Molecular and cellular biology, 7(2), 725–737. https://doi.org/10.1128/mcb.7.2.725
  12. ↑ de Wet, J. R., Wood, K. V., Helinski, D. R., & DeLuca, M. (1985). Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 82(23), 7870–7873. https://doi.org/10.1073/pnas.82.23.7870
  13. ↑ Thorne, N., Shen, M., Lea, W. A., Simeonov, A., Lovell, S., Auld, D. S., & Inglese, J. (2012). Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Chemistry & biology, 19(8), 1060–1072. https://doi.org/10.1016/j.chembiol.2012.07.015

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