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Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate

Native Oplophorus luciferase (OpLase) and its catalytic 19 kDa protein (wild KAZ) show highest luminescence activity with coelenterazine (CTZ) among CTZ analogs. Mutated wild KAZ with 16 amino acid substitutions (nanoKAZ/nanoLuc) utilizes bis-coelenterazine (bis-CTZ) as the preferred substrate and e...

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Autores principales: Inouye, Satoshi, Sato, Jun-ichi, Sahara-Miura, Yuiko, Tomabechi, Yuri, Sumida, Yuto, Sekine, Shun-ichi, Shirouzu, Mikako, Hosoya, Takamitsu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9491549/
https://www.ncbi.nlm.nih.gov/pubmed/36129943
http://dx.doi.org/10.1371/journal.pone.0272992
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author Inouye, Satoshi
Sato, Jun-ichi
Sahara-Miura, Yuiko
Tomabechi, Yuri
Sumida, Yuto
Sekine, Shun-ichi
Shirouzu, Mikako
Hosoya, Takamitsu
author_facet Inouye, Satoshi
Sato, Jun-ichi
Sahara-Miura, Yuiko
Tomabechi, Yuri
Sumida, Yuto
Sekine, Shun-ichi
Shirouzu, Mikako
Hosoya, Takamitsu
author_sort Inouye, Satoshi
collection PubMed
description Native Oplophorus luciferase (OpLase) and its catalytic 19 kDa protein (wild KAZ) show highest luminescence activity with coelenterazine (CTZ) among CTZ analogs. Mutated wild KAZ with 16 amino acid substitutions (nanoKAZ/nanoLuc) utilizes bis-coelenterazine (bis-CTZ) as the preferred substrate and exhibits over 10-fold higher maximum intensity than CTZ. To understand the substrate selectivity of nanoKAZ between CTZ and bis-CTZ, we prepared the reverse mutants of nanoKAZ by amino acid replacements with the original amino acid residue of wild KAZ. The reverse mutant with L18Q and V27L substitutions (QL-nanoKAZ) exhibited 2.6-fold higher maximum intensity with CTZ than that of nanoKAZ with bis-CTZ. The catalytic properties of QL-nanoKAZ including substrate specificity, luminescence spectrum, luminescence kinetics, luminescence products of CTZ, and luminescence inhibition by deaza-CTZ analogs were characterized and were compared with other CTZ-utilizing luciferases such as Gaussia and Renilla luciferases. Thus, QL-nanoKAZ with CTZ could be used as a potential reporter protein for various luminescence assay systems. Furthermore, the crystal structure of QL-nanoKAZ was determined at 1.70 Å resolution. The reverse mutation at the L18Q and V27L positions of α2-helix in nanoKAZ led to changes in the local structures of the α4-helix and the β6- and β7-sheets, and might enhance its binding affinity and oxidation efficiency with CTZ to emit light.
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spelling pubmed-94915492022-09-22 Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate Inouye, Satoshi Sato, Jun-ichi Sahara-Miura, Yuiko Tomabechi, Yuri Sumida, Yuto Sekine, Shun-ichi Shirouzu, Mikako Hosoya, Takamitsu PLoS One Research Article Native Oplophorus luciferase (OpLase) and its catalytic 19 kDa protein (wild KAZ) show highest luminescence activity with coelenterazine (CTZ) among CTZ analogs. Mutated wild KAZ with 16 amino acid substitutions (nanoKAZ/nanoLuc) utilizes bis-coelenterazine (bis-CTZ) as the preferred substrate and exhibits over 10-fold higher maximum intensity than CTZ. To understand the substrate selectivity of nanoKAZ between CTZ and bis-CTZ, we prepared the reverse mutants of nanoKAZ by amino acid replacements with the original amino acid residue of wild KAZ. The reverse mutant with L18Q and V27L substitutions (QL-nanoKAZ) exhibited 2.6-fold higher maximum intensity with CTZ than that of nanoKAZ with bis-CTZ. The catalytic properties of QL-nanoKAZ including substrate specificity, luminescence spectrum, luminescence kinetics, luminescence products of CTZ, and luminescence inhibition by deaza-CTZ analogs were characterized and were compared with other CTZ-utilizing luciferases such as Gaussia and Renilla luciferases. Thus, QL-nanoKAZ with CTZ could be used as a potential reporter protein for various luminescence assay systems. Furthermore, the crystal structure of QL-nanoKAZ was determined at 1.70 Å resolution. The reverse mutation at the L18Q and V27L positions of α2-helix in nanoKAZ led to changes in the local structures of the α4-helix and the β6- and β7-sheets, and might enhance its binding affinity and oxidation efficiency with CTZ to emit light. Public Library of Science 2022-09-21 /pmc/articles/PMC9491549/ /pubmed/36129943 http://dx.doi.org/10.1371/journal.pone.0272992 Text en © 2022 Inouye et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Inouye, Satoshi
Sato, Jun-ichi
Sahara-Miura, Yuiko
Tomabechi, Yuri
Sumida, Yuto
Sekine, Shun-ichi
Shirouzu, Mikako
Hosoya, Takamitsu
Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate
title Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate
title_full Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate
title_fullStr Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate
title_full_unstemmed Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate
title_short Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate
title_sort reverse mutants of the catalytic 19 kda mutant protein (nanokaz/nanoluc) from oplophorus luciferase with coelenterazine as preferred substrate
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9491549/
https://www.ncbi.nlm.nih.gov/pubmed/36129943
http://dx.doi.org/10.1371/journal.pone.0272992
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