7VSX

Crystal structure of QL-nanoKAZ (Reverse mutant of nanoKAZ with L18Q and V27L)

7VSX の概要

• エントリーDOI: 10.2210/pdb7vsx/pdb
• 分子名称: QLnK, 2-(N-MORPHOLINO)-ETHANESULFONIC ACID (3 entities in total)
• 機能のキーワード: mutated 19 kda protein, oplophorus luciferase, luminescent protein
• 由来する生物種: Oplophorus gracilirostris
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 19383.09

構造登録者

Tomabechi, Y.,Sekine, S.,Shirouzu, M.,Takamitsu, H.,Satoshi, I. (登録日: 2021-10-27, 公開日: 2022-08-24, 最終更新日: 2023-11-29)

主引用文献

Inouye, S.,Sato, J.I.,Sahara-Miura, Y.,Tomabechi, Y.,Sumida, Y.,Sekine, S.I.,Shirouzu, M.,Hosoya, T.
Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate.
Plos One, 17:e0272992-e0272992, 2022

PubMed Abstract: 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.

PubMed: 36129943
DOI: 10.1371/journal.pone.0272992

実験手法

X-RAY DIFFRACTION (1.698 Å)