User:Matt Whelihan: Difference between revisions

Line 10:

Both the Japanese and North American luciferase variants are single-chain 62 kDa monooxygenases with two distinct domains. Their <scene name='User:Matt_Whelihan/Secondary_structure/1'>structures</scene> are comprised of a mix of <font color='magenta'>alpha</font> and <font color='gold'>beta</font> secondary structures that form four distinct motifs. The <scene name='User:Matt_Whelihan/N-terminal_domain/1'>N-terminal domain</scene> (residues 4-436) is comprised of of an antiparalell β-barrel and two β-sheets, flanked by α-helices which forms a typical Rossmann fold [http://images.google.com/imgres?imgurl=http://chem.csusb.edu/~dpedersn/C436/openbetarossman.jpg&imgrefurl=http://chem.csusb.edu/~dpedersn/C436/prot_struct_and_class.html&usg=__xOF4ifmAyJ5qm3UPaNKd9whKlu4=&h=539&w=719&sz=35&hl=en&start=5&um=1&tbnid=IMkWKmy0xzWcYM:&tbnh=105&tbnw=140&prev=/images%3Fq%3DRossman%2BFold%26hl%3Den%26safe%3Doff%26sa%3DN%26um%3D1] The

<scene name='User:Matt_Whelihan/C-terminal_domain/2'>C-terminal domain</scene> (residues 440-544) is comprises of a separate α-β hinge<ref>PMID:8805533</ref>. Firefly luciferases share significant sequence and mechanistic homology with peptide synthetases and acylCoA ligases. These enzymes belong to a superfamily of adenylate-forming enzymes that catalyze activation reactions between ATP and a carboxyl group of their substrates. This group of proteins shares an identifying motif <scene name='User:Matt_Whelihan/198-207/1'>(198SerSerGlySerThrGlyLeuProLysGly207)</scene> and has been termed the “acyl-adenylate/thioester-forming” enzyme family. Despite high sequence homology, there are only seven residues that are conserved across this superfamily (Gly200, Lys206, Glu344, Asp422, Arg437, Gly446, and Glu455). These residues are believed to be integral to the binding of ATP and the formation of an adenylate compound <ref>PMID:1351742</ref><ref>PMID:1447981</ref>. These residues however are located across the N and C-terminal domains, which in the structure, are too far apart to produce catalysis. This suggested suggest that the crystallized form was in the resting state of the enzyme and it was hypothesized that the C-terminal domain would close in on the active site cleft upon substrate binding. This closing of the active site combined with various hydrophobic residues seen packed around the active site suggested that catalysis may occur in the absence of water.

These seven highly conserved amino acids identify the <scene name='User:Matt_Whelihan/Active_site_residues/1'>active site</scene> which is located in the large hydrophobic cleft between the two adjacent N and C-terminal domains.

These seven highly conserved amino acids identify the <scene name='User:Matt_Whelihan/Active_site_residues/1'>active site</scene> which is located in the large hydrophobic cleft between the two adjacent N and C-terminal domains. [[Media:Example.ogg]]

Firefly luciferases catalyze the formation of the luciferin-adenylate intermediate. A proton is then abstracted in a steriospecific manner from the C-4 carbon, presumably by an enzyme base. A conformational change then occurs which allows molecular oxygen addition to the newly formed anion. As the highly reactive dioxetanone intermediate decays to the ground state, it releases a photon of light<ref>PMID:7000855</ref><ref>PMID:4813372</ref>. One of the most intriguing parts of this mechanism is about how various firefly species are able to emit different types of light with the same exact reaction. It had been hypothesized for 20 years that the enol form was responsible for the typical yellow green spectra at neutral pH by an enzyme assisted tautomerization. The keto form of the intermediate which is seen at low pH gave rise to the red spectra, presumably due to a less stable charge transfer leading to a less efficient conversion into the lower energy red Various mutational studies by Branchini et al. showed that seven residues <scene name='User:Matt_Whelihan/Red_shift/1'>His245, Phe247, Arg218, Ala348, Gly341, Asp422 and Thr343</scene> were responsible for controlling the resonance-based charge delocalization of the anionic keto form of oxiluciferin ~~light~~<ref>PMID:15182171</ref>.

Firefly luciferases catalyze the formation of the luciferin-adenylate intermediate. A proton is then abstracted in a steriospecific manner from the C-4 carbon, presumably by an enzyme base. A conformational change then occurs which allows molecular oxygen addition to the newly formed anion. As the highly reactive dioxetanone intermediate decays to the ground state, it releases a photon of light<ref>PMID:7000855</ref><ref>PMID:4813372</ref>.

One of the most intriguing parts of this mechanism is about how various firefly species are able to emit different types of light with the same exact reaction. It had been hypothesized for 20 years that the enol form was responsible for the typical yellow green spectra at neutral pH by an enzyme assisted tautomerization. The keto form of the intermediate which is seen at low pH gave rise to the red spectra, presumably due to a less stable charge transfer leading to a less efficient conversion into the lower energy red Various mutational studies by Branchini et al. showed that seven residues <scene name='User:Matt_Whelihan/Red_shift/1'>His245, Phe247, Arg218, Ala348, Gly341, Asp422 and Thr343</scene> were responsible for controlling the resonance-based charge delocalization of the anionic keto form of oxiluciferin <ref>PMID:15182171</ref>. This control of resonance charge on the molecule is paramount in the enzymes ability to catalyze the yellow green emission of light following oxidation of the substrate. Various species have evolved with subtle variations in their active site structures which in turn yields a large spectral shift as the keto form of the oxyluciferin is favored.

----

User:Matt Whelihan: Difference between revisions

Navigation menu

Page actions

Page actions

Personal tools

Navigation

Search

Tools

@@ Line 10: / Line 10: @@
 Both the Japanese and North American luciferase variants are single-chain 62 kDa monooxygenases with two distinct domains. Their  <scene name='User:Matt_Whelihan/Secondary_structure/1'>structures</scene> are comprised of a mix of <font color='magenta'>alpha</font> and <font color='gold'>beta</font> secondary structures that form four distinct motifs. The <scene name='User:Matt_Whelihan/N-terminal_domain/1'>N-terminal domain</scene> (residues 4-436) is comprised of of an antiparalell β-barrel and two β-sheets, flanked by α-helices which forms a typical Rossmann fold [http://images.google.com/imgres?imgurl=http://chem.csusb.edu/~dpedersn/C436/openbetarossman.jpg&imgrefurl=http://chem.csusb.edu/~dpedersn/C436/prot_struct_and_class.html&usg=__xOF4ifmAyJ5qm3UPaNKd9whKlu4=&h=539&w=719&sz=35&hl=en&start=5&um=1&tbnid=IMkWKmy0xzWcYM:&tbnh=105&tbnw=140&prev=/images%3Fq%3DRossman%2BFold%26hl%3Den%26safe%3Doff%26sa%3DN%26um%3D1] The
 <scene name='User:Matt_Whelihan/C-terminal_domain/2'>C-terminal domain</scene> (residues 440-544) is comprises of a separate α-β hinge<ref>PMID:8805533</ref>. Firefly luciferases share significant sequence and mechanistic homology with peptide synthetases and acylCoA ligases. These enzymes belong to a superfamily of adenylate-forming enzymes that catalyze activation reactions between ATP and a carboxyl group of their substrates. This group of proteins shares an identifying motif <scene name='User:Matt_Whelihan/198-207/1'>(198SerSerGlySerThrGlyLeuProLysGly207)</scene> and has been termed the “acyl-adenylate/thioester-forming” enzyme family. Despite high sequence homology, there are only seven residues that are conserved across this superfamily (Gly200, Lys206, Glu344, Asp422, Arg437, Gly446, and Glu455). These residues are believed to be integral to the binding of ATP and the formation of an adenylate compound <ref>PMID:1351742</ref><ref>PMID:1447981</ref>. These residues however are located across the N and C-terminal domains, which in the structure, are too far apart to produce catalysis. This suggested suggest that the crystallized form was in the resting state of the enzyme and it was hypothesized that the C-terminal domain would close in on the active site cleft upon substrate binding. This closing of the active site combined with various hydrophobic residues seen packed around the active site suggested that catalysis may occur in the absence of water.
-These seven highly conserved amino acids identify the <scene name='User:Matt_Whelihan/Active_site_residues/1'>active site</scene> which is located in the large hydrophobic cleft between the two adjacent N and C-terminal domains.
+These seven highly conserved amino acids identify the <scene name='User:Matt_Whelihan/Active_site_residues/1'>active site</scene> which is located in the large hydrophobic cleft between the two adjacent N and C-terminal domains. [[Media:Example.ogg]]
-Firefly luciferases catalyze the formation of the luciferin-adenylate intermediate. A proton is then abstracted in a steriospecific manner from the C-4 carbon, presumably by an enzyme base. A conformational change then occurs which allows molecular oxygen addition to the newly formed anion. As the highly reactive dioxetanone intermediate decays to the ground state, it releases a photon of light<ref>PMID:7000855</ref><ref>PMID:4813372</ref>. One of the most intriguing parts of this mechanism is about how various firefly species are able to emit different types of light with the same exact reaction. It had been hypothesized for 20 years that the enol form was responsible for the typical yellow green spectra at neutral pH by an enzyme assisted tautomerization. The keto form of the intermediate which is seen at low pH gave rise to the red spectra, presumably due to a less stable charge transfer leading to a less efficient conversion into the lower energy red Various mutational studies by Branchini et al.  showed that seven residues <scene name='User:Matt_Whelihan/Red_shift/1'>His245, Phe247, Arg218, Ala348, Gly341, Asp422 and Thr343</scene> were responsible for controlling the resonance-based charge delocalization of the anionic keto form of oxiluciferin light<ref>PMID:15182171</ref>.
+Firefly luciferases catalyze the formation of the luciferin-adenylate intermediate. A proton is then abstracted in a steriospecific manner from the C-4 carbon, presumably by an enzyme base. A conformational change then occurs which allows molecular oxygen addition to the newly formed anion. As the highly reactive dioxetanone intermediate decays to the ground state, it releases a photon of light<ref>PMID:7000855</ref><ref>PMID:4813372</ref>.
+One of the most intriguing parts of this mechanism is about how various firefly species are able to emit different types of light with the same exact reaction. It had been hypothesized for 20 years that the enol form was responsible for the typical yellow green spectra at neutral pH by an enzyme assisted tautomerization. The keto form of the intermediate which is seen at low pH gave rise to the red spectra, presumably due to a less stable charge transfer leading to a less efficient conversion into the lower energy red Various mutational studies by Branchini et al.  showed that seven residues <scene name='User:Matt_Whelihan/Red_shift/1'>His245, Phe247, Arg218, Ala348, Gly341, Asp422 and Thr343</scene> were responsible for controlling the resonance-based charge delocalization of the anionic keto form of oxiluciferin <ref>PMID:15182171</ref>. This control of resonance charge on the molecule is paramount in the enzymes ability to catalyze the yellow green emission of light following oxidation of the substrate. Various species have evolved with subtle variations in their active site structures which in turn yields a large spectral shift as the keto form of the oxyluciferin is favored.
 ----