Biology subjects

Sakuma, M.

Publications and source records attributed to Sakuma, M..

9 recordsLinked to original sources

bioRxiv2026

Osmotic-stress-inducible nuclear condensates restrict gene inducibility

Plants, as sessile organisms, have developed various mechanisms to respond to environmental stress conditions. The plant hormone abscisic acid (ABA) is necessary for the plant to adapt to osmotic stress conditions. However, the molecular mechanisms preceding ABA accumulation remain largely unknown. To isolate transcriptional complexes on the promoter region of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) encoding a rate-limiting enzyme in the ABA biosynthetic pathway in planta, we developed the insertional chromatin immunoprecipitation (iChIP) screen method. The identified ALBA proteins formed condensates through liquid-liquid phase separation (LLPS) in response to osmotic stress conditions. ALBA4 directly binds to stress-inducible genes, including NCED3, and suppresses their stress inducibility. Our results demonstrate how plants respond to osmotic stress at early timepoints before ABA biosynthesis through condensate formation as osmo-sensors.

plant biology↗

bioRxiv2025

Triplet Quenching by Active Site Cysteine Residues Improves Photostability in Fatty Acid Photodecarboxylase

Enzyme photobiocatalysis uses light to drive high-energy transformations but is limited by the rarity of photoenzymes. Fatty acid photodecarboxylase (FAP), a recently discovered photoenzyme, enables fatty acid conversion to alkanes/alkenes via excitation of an FAD cofactor, though its poor photostability and photoinactivation has hindered industrial applications. Here, we combine protein engineering approaches with biocatalytic and biophysical techniques, as well as computational chemistry, to demonstrate that additional active site cysteine residues can suppress oxygen-mediated inactivation processes that are driven by the FAD triplet-excited state. We identify a number of positions close to the FAD for cysteine residues that lead to a significant enhancement in activity as a result of an increase in the number of catalytic turnovers and improved photostability. The additional cysteine residues quench the triplet excited state of the FAD cofactor via a proposed proton-coupled electron transfer mechanism, resulting in lower levels of harmful reactive oxygen species. Our study highlights promising routes to mitigate non-productive, photoinactivation pathways in FAP and informs the rational design of new flavin-based photoenzymes.

biochemistry↗

bioRxiv2025

Integrated structural dynamics uncover new modes of B12 photoreceptor activation

Photoreceptor proteins initiate, regulate and control fundamental biological processes such as vision, photosynthesis and circadian rhythms1. A large photoreceptor subfamily uses vitamin B12 derivatives for light sensing2, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions3. The molecular mechanism of B12 photoreception and how this differs to the thermal pathways remain unknown. Here we provide a detailed spatio-temporal description of photoactivation in the prototypical tetrameric B12 photoreceptor CarH4,5 from nanoseconds to seconds by using an integrative approach, combining time- and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. High resolution structural snapshots of key intermediates illustrate how photocleavage of a Co-C bond triggers a pathway of structural changes that propagate throughout CarH from the B12 chromophore, via a previously unknown adduct, to finally cause tetramer dissociation. These unique intermediates, which differentiate CarH from thermally-activated B12 enzymes, steer the photoactivation pathway and act as the molecular bridge between photochemical and photobiological timescales. Our results offer a spatio-temporal understanding of CarH photoactivation and pave the way for designing and optimising B12-dependent photoreceptors for future optogenetic applications.

biophysics↗

bioRxiv2025

Widespread promiscuous alkaline phosphatases underscore early microbial phosphite utilization

Phosphate is often a limiting resource, directly affecting the availability of key biomolecules such as nucleotides. To cope with phosphate scarcity, bacteria have evolved enzymes that utilize alternative phosphorus compounds, including phosphite (Pt). Although a few enzymes oxidize Pt to produce phosphate, the enzymes responsible for Pt oxidation in many environmental bacteria remain unidentified, and the role of microbial Pt oxidation in the global phosphorus cycle is not yet fully understood. In this study, we performed bioinformatic analyses of three Pt-oxidizing enzymes: the native Pt oxidase phosphite dehydrogenase (PtxD), and two promiscuous Pt oxidases, alkaline phosphatase (AP) and carbon-phosphorus (CP) lyase. Among these, AP was found to be widely distributed across bacteria since the early stages of their evolution. In contrast, PtxD emerged later in a limited number of bacterial lineages that had lost AP. Our biochemical characterizations revealed that most extant and reconstructed ancestral APs tested exhibited Pt oxidation activity. Moreover, disruption of active-site residues diminished Pt oxidase activity in AP, while only partially affecting its native function. This promiscuous function of AP reveals an overlooked mechanism in bacterial phosphate metabolism and underscores the role of Pt in the cycling of bioavailable phosphorus in ecosystems.

biochemistry↗

bioRxiv2025

Single-Molecule Kinetic Exploration of Functional Substates in an Evolving Phosphotriesterase

Enzymes achieve catalysis by dynamically sampling diverse conformational states. Beyond this plasticity, individual enzyme molecules occupy metastable substates, forming an ensemble of functional substates within a population. Since shifts in functional substate dynamics drive phenotypic variation, their evolutionary trajectories are central to the emergence of new functions. However, the challenge of measuring functional substates has hindered our understanding of their role in enzyme evolution and the optimization of conformational substates. Here, we address this gap by investigating how functional and conformational substates were modulated during enzyme functional transitions, using single-molecule kinetic assays and molecular dynamics simulations. We analyzed wild-type phosphotriesterase (PTE) and 18 evolved variants that transitioned from the native PTE to promiscuous arylesterase (AE) activity. Our findings reveal that evolutionary transitions reshape functional and conformational substate landscapes: PTE-specialized variants exhibit broader substate distributions, whereas AE-specialized variants display more uniform substates. These results provide the first direct evidence that enzyme evolution is accompanied by coordinated shifts in functional and conformational substate equilibria, optimizing both for the enzymes catalytic efficiency. This work highlights the power of single-molecule techniques in uncovering how heterogeneous enzyme populations navigate substate transitions and, ultimately, how these transitions shape enzyme evolvability.

biochemistry↗

bioRxiv2023

Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase.

The photoenzyme protochlorophyliide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide (Pchlide) to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight about the light-driven reaction chemistry, a structural rationale for POR photocatalysis has proved more challenging and remains hotly debated. Recent structural models of the ternary enzyme-substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the Pchlide substrate and the architecture of the POR active site that have significant implications for the catalytic mechanism of Pchlide reduction. Here, we have used a combination of computational and experimental approaches to investigate the compatibility of each of these structural models with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR-Pchlide-NADPH ternary complex based on these findings. Through detailed site-directed mutagenesis studies we show that a strictly conserved Tyr residue, which has previously been proposed to act as the proton donor in POR photocatalysis, is not likely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site Cys residue is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water-mediated network. Moreover, a conserved Gln residue is found to be important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal binding pose for the POR ternary enzyme-substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme.

biochemistry↗

bioRxiv2021

Genetic perturbation enhances functional heterogeneity in alkaline phosphatase

Enzymes inherently exhibit molecule-to-molecule heterogeneity in catalytic activity or function, which underlies the acquisition of new functions in evolutionary processes. However, correlations between the functional heterogeneity of an enzyme and its multi-functionality or promiscuity remain elusive. In addition, the modulation of functional heterogeneity upon genetic perturbation is currently unexplored. Here, we quantitatively analyzed functional heterogeneity in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase (AP) by employing single-molecule assay with a femtoliter reactor array device. Most mutant enzymes exhibited higher functional heterogeneity than the wild-type enzyme, irrespective of catalytic activity. These results indicated that the wild-type AP minimizes functional heterogeneity, and single-point mutations can significantly expand the span of functional heterogeneity in AP. Moreover, we identified a clear correlation between functional heterogeneity and promiscuous activities. These findings suggest that enzymes can acquire greater functional heterogeneity following marginal genetic perturbations that concomitantly promote catalytic promiscuity.

biochemistry↗

bioRxiv2019

Quantitative Detection of Cell Activity by Measuring the Fluctuation of Intracellular Motility

The measurement of cell activity changes during damage is important to understand the process of cell death and evaluate the effect of medicines. To evaluate cell activity generally, we extended the method of intensity fluctuation in which intensity change in the pixel induced by the movement of organelles was calculated. Cancer, endothelial and iPS cells were damaged by reactive oxygen species (ROS) generated by a fluorescent dye (IR700), hydrogen peroxide, and ultraviolet light. The intensity fluctuation in damaged cells gradually decreased independent of the kind of cell, indicating that the decrease in the fluctuation is a general phenomenon in damaged cells. The rupture of vesicles and mitochondria in the cells were observed upon ROS production. The motility of purified kinesin and dynein which transport vesicles and organelles was inhibited by ROS. These suggest that ROS and cytotoxic molecules spreading from ruptured organelles contribute to the reduction in cell activity which brings about the decrease in the motility and intensity fluctuation of organelles driven by kinesin and dynein.

cell biology↗

bioRxiv2018

Rac-dependent signaling from keratinocytes promotes differentiation of intradermal white adipocytes

Rac signaling affects numerous downstream targets; however, few studies have established in vivo levels. We generated mice with a single knockout (KO) of Rac1 (Keratin5 (K5)-Cre;Rac1flox/flox, Rac1-KO) and double KO of Rac1 and Rac3 (K5-Cre;Rac1flox/flox;Rac3-/-, Rac1/Rac3-DKO) in keratinocytes. Strikingly, Rac1-KO mice exhibited thinner dermal white adipose tissue, which was considerably further reduced in Rac1/Rac3-DKO mice. DNA microarray using primary keratinocytes from Rac1/Rac3-DKO mice exhibited decreased mRNA levels of Bmp2, Bmp5, Fgf20, Fgf21, Fgfbp1, and Pdgf. Combinational treatment with BMP2 and FGF21 or BMP2 and FGF20 in culture medium, but not individual purified recombinant proteins, could differentiate 3T3-L1 fibroblasts into adipocytes, as could culture media obtained from primary keratinocytes. Conversely, addition of anti-BMP2 or anti-FGF21 antibodies into the culture medium inhibited fibroblast differentiation. Furthermore, combinational treatment with BMP2 and FGF21 promoted adipocyte differentiation only of rat primary white, but not brown, adipocyte precursors. Notably, brown adipogenesis by FGF21 was inhibited by BMP2. Thus, we proposed novel paracrine pathways from keratinocytes to intradermal pre-adipocytes, which function as Rac-dependent modulators of white adipogenesis, but also brown adipogenesis.

cell biology↗