Biology subjects

Chaiyen, P.

Publications and source records attributed to Chaiyen, P..

5 recordsLinked to original sources

bioRxiv2025

Kinetic mechanism of Renilla luciferase guides induced-fit engineering for improved bioluminescence

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 work establishes a mechanistically grounded framework for the development of next-generation bioluminescent tools and for engineering enzymes controlled by dynamically gated ligand exchange.

biochemistry↗

bioRxiv2025

Thermodynamically Explicit Kinetics with Potential for Genome-wide Application

Metabolic processes are inherently dynamic, yet genome-scale models are often limited to steady-state analysis, which cannot capture time-dependent responses crucial for understanding complex diseases like cancer. This work introduces a Thermodynamically Explicit (TDE) kinetic framework to bridge this gap by constructing dynamic metabolic models grounded in fundamental thermodynamic principles. Our approach uses standard chemical potentials to derive simplified rate laws for metabolic reactions, reducing the kinetic complexity of each reaction to a single, biochemically determined prefactor ({lambda}), which we will name kinetic capacity factor. The number of parameters in the suggested TDE kinetics is indeed minimal in the sense that it uses the lowest possible number of free parameters required to define a reaction rate that is both dynamic and thermodynamically consistent. We demonstrate the validity and practical application of this framework by re-parameterizing a well-established kinetic model of glycolysis. The resulting TDE model successfully reproduces the dynamic behavior of the original, more complex model in simulations. By streamlining the parameterization process, the TDE kinetic framework offers a powerful and scalable tool for building genome-wide dynamic metabolic models. This approach paves the way for more accurate and predictive simulations of metabolic behavior, with significant potential for applications in systems biology, medicine, and biotechnology.

biophysics↗

bioRxiv2023

A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase levels of a pool of sugar-phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells; demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase, GDH) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology. TeaserUse of sugar and sugar reductase to increase sugar phosphates for enhancing in situ synthesis of cofactors upon cellular demand for synthetic biology.

synthetic biology↗

bioRxiv2022

A catalytic mechanism for Renilla-type bioluminescence

The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type bioluminescence through crystallographic, spectroscopic, and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing unprecedented snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O2 via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase-luciferin pairs for ultrasensitive optical bioassays.

biochemistry↗

bioRxiv2021

High Sensitivity and Low-Cost Flavin luciferase (FLUX)-based Reporter Gene for Mammalian Cell Expression

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most common, and thus most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase for Mammalian Cell Expression (FLUX) by engineering luciferase from Vibrio campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells. We found that the FLUX reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUX/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We demonstrated that FLUX can be used for testing inhibitors of the NF-{kappa}B signaling pathway, validating FLUX applications for various assays in the future.

biochemistry↗