Conformational gating of product release sets the catalytic ceiling of a bioluminescent reporter
Toul, M.; Horackova, J.; Schenkmayerova, A.; Planas-Iglesias, J.; Landolt, T.; Sucharitakul, J.; Janin, Y.; Prakinee, K.; Chaiyen, P.; Stavrakis, S.; deMello, A.; Johnson, K. A.; Damborsky, J.; Marek, · biochemistry · 2026-09-07 · 原文
DOI：10.1101/2025.09.16.675553作者：14 位
Luciferases are widely used bioluminescent reporters, yet the molecular determinants of their catalytic efficiency and light-emission stability remain incompletely understood. Here, we reconstruct the complete catalytic pathway of Renilla luciferase by combining steady-state, transient, and temperature-dependent kinetics with crystallography and molecular simulations. We show that the enzyme is substantially undersaturated with oxygen (Km,O2 = 719 M), causing its true turnover number (kcat = 21.9 s-1) to be systematically underestimated. Concurrently, elevated oxygen drives irreversible enzyme inactivation after ~1,500 turnovers, revealing a fundamental trade-off that limits oxygen-affinity engineering. Instead, the genuine bottleneck of the catalytic cycle is the induced-fit conformational opening of the product-bound enzyme. Selective engineering of this transition step through rational loop grafting yielded a variant AncFT-L14 with enhanced catalytic efficiency and glow-type bioluminescence with substantially slower signal decay in cell lysates. Collectively, our results identify conformational dynamics as a primary tunable determinant of luciferase function. More broadly, this
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1. 人话版
Luciferases are widely used bioluminescent reporters, yet the molecular determinants of their catalytic efficiency and light-emission stability remain incompletely understood.
Here, we reconstruct the complete catalytic pathway of Renilla luciferase by combining steady-state, transient, and temperature-dependent kinetics with crystallography and molecular simulations.
2. 领域脉络
本文类目：biochemistry，属于其所在研究脉络的最新进展。
3. 机制拆解
Instead, the genuine bottleneck of the catalytic cycle is the induced-fit conformational opening of the product-bound enzyme.
4. 证据与数字
We show that the enzyme is substantially undersaturated with oxygen (Km,O2 = 719 M), causing its true turnover number (kcat = 21.9 s-1) to be systematically underestimated.
Concurrently, elevated oxygen drives irreversible enzyme inactivation after ~1,500 turnovers, revealing a fundamental trade-off that limits oxygen-affinity engineering.
Selective engineering of this transition step through rational loop grafting yielded a variant AncFT-L14 with enhanced catalytic efficiency and glow-type bioluminescence with substantially slower signal decay in cell lysates.
5. 反例与边界
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6. 跨领域连接与意外收获
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7. 可复用方法
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8. 术语表
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