A suicidal and extensively disordered luciferase with a bright luminescence
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A suicidal and extensively disordered luciferase with a bright luminescence. / Dijkema, Fenne Marjolein; Escarpizo-Lorenzana, Marta Iglesia; Nordentoft, Matilde Knapkøien; Rabe, Hanna Christin; Sahin, Cagla; Landreh, Michael; Branca, Rui Mamede; Sørensen, Esben Skipper; Christensen, Brian; Prestel, Andreas; Teilum, Kaare; Winther, Jakob Rahr.
In: Protein Science, Vol. 33, No. 8, e5115, 2024.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - A suicidal and extensively disordered luciferase with a bright luminescence
AU - Dijkema, Fenne Marjolein
AU - Escarpizo-Lorenzana, Marta Iglesia
AU - Nordentoft, Matilde Knapkøien
AU - Rabe, Hanna Christin
AU - Sahin, Cagla
AU - Landreh, Michael
AU - Branca, Rui Mamede
AU - Sørensen, Esben Skipper
AU - Christensen, Brian
AU - Prestel, Andreas
AU - Teilum, Kaare
AU - Winther, Jakob Rahr
N1 - Publisher Copyright: © 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
PY - 2024
Y1 - 2024
N2 - Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.
AB - Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.
KW - active site
KW - enzyme
KW - intrinsic disorder
KW - luciferase
KW - NMR
KW - oxidation
KW - structure
U2 - 10.1002/pro.5115
DO - 10.1002/pro.5115
M3 - Journal article
AN - SCOPUS:85198725705
VL - 33
JO - Protein Science
JF - Protein Science
SN - 0961-8368
IS - 8
M1 - e5115
ER -
ID: 399109309