Biology subjects

Komatsu, K.

Publications and source records attributed to Komatsu, K..

17 recordsLinked to original sources

bioRxiv2026

Proximity Labeling-Assisted Flow Cytometry Improves Fluorescence Contrast in Extracellular Vesicle-Enriched Fractions

Extracellular vesicles (EVs) are potential sources of circulating biomarkers, but disease-associated EVs are often scarce and their nanoscale dimensions limit analysis by conventional flow cytometry. To improve the detection of disease-associated EV signals using widely available instruments, we developed a workflow combining preparation of serum EV-enriched fractions by size-exclusion chromatography, CHL1-initiated enzyme-mediated activation of radical sources (EMARS) proximity labeling, and conventional flow cytometry. EMARS deposits fluorescein on molecules proximal to antibody-bound CHL1, thereby increasing target-initiated fluorescence. In a representative comparison, EMARS increased the signal-to-background ratio from 1.21 with conventional antibody labeling to 6.28. Because unlabeled EVs and debris interfered with analysis, we used an AI-assisted procedure to explore FITC-H cutoffs for retained P1 events. ChatGPT compared 65 candidate cutoffs across statistical and event-retention metrics and recommended 1000; the authors reviewed the complete results before adopting this value. At this data-selected cutoff, mean fluorescence intensity was higher in interstitial lung disease (ILD) than in non-ILD samples among 28 evaluable samples (P = 0.002; area under the receiver operating characteristic curve [AUC] = 0.846); neither value was adjusted for cutoff selection. Post hoc nested leave-oneout cross-validation (LOOCV), in which cutoff selection was repeated within each outer fold, yielded an AUC of 0.716 with 29 of 34 samples evaluable. The association with ILD requires validation in independent cohorts, but these exploratory findings suggest that proximity labeling may enhance measurable fluorescence contrast in serum EV-enriched fractions and support further evaluation of disease-associated EV signals using conventional flow cytometers.

biochemistry↗

bioRxiv2026

Comprehensive functional mapping of accessory chromosomes identifies a dominant virulence-regulator paralog in tomato wilt pathogen

Accessory chromosomes (ACs) serve as flexible genomic compartments that facilitate rapid adaptive evolution in eukaryotic microbes. In the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici, ACs are essential for virulence and host specificity; however, their structural complexity and functional redundancy have hindered a systematic characterization of their distinct roles. Here, we established a CRISPR/Cas9-based chromosomal dissection platform to generate a comprehensive functional map of pathogenicity determinants within ACs. Using this platform, we successfully generated a library of 36 large deletion mutants across the approximately 10 Mb putative AC region, enabling a chromosome-scale functional characterization of these compartments. A systematic screen of this library identified seven discrete AC segments that are indispensable for full virulence toward tomato. High-resolution chromosomal dissection of one virulence-associated segment through iterative subdivision and targeted gene disruption revealed that FTF1a-1, a single member of the Fusarium transcription factor 1 (FTF1) family that originated from the duplication of core-chromosomal virulence gene FTF2 into the AC region, functions as a dominant regulator of virulence. Given that the FTF1a-3 paralog contributes only marginally to virulence and the deletion of other paralogs does not markedly affect disease development, our findings demonstrate a functional hierarchy within the duplicated FTF1 gene family. These results imply that neofunctionalization of virulence genes within plastic fungal genomes promotes hyper-virulence and drives host-specific adaptation in F. oxysporum.

microbiology↗

bioRxiv2026

A single-stranded DNA virus replicates in the mitochondria of a marine oomycete

A virus with a circular single-stranded DNA genome, HthCRESSV1, was discovered in an oomycete, Halophytophthora thermoambigua, isolated from brackish waters off the Algarve coast in southern Portugal. Phylogenetic analyses place this virus outside of all currently classified families within the phylum Cressdnaviricota, suggesting it represents a distinct lineage. Cellular fractionation, mitochondrial marker co-enrichment, and Southern blot analyses indicate that HthCRESSV1 is associated with mitochondria and replicates episomally. The virus is stably transmitted through zoospores and, notably, infected host isolates are associated with reduced growth and changes in temperature-dependent performance. Homologous sequences corresponding to both the replication-associated protein and a membrane-associated hypothetical protein were identified in the mitochondrial genomes of several oomycete species, suggesting recurrent endogenization of HthCRESSV1-like viruses in oomycete mitochondria. Together, these findings expand the known diversity of oomycete-associated DNA viruses, identify mitochondria as an unexpected niche for DNA virus replication, and highlight marine environments as underexplored reservoirs of DNA viruses in stramenopiles.

evolutionary biology↗

bioRxiv2026

Comprehensive transcriptomic and proteomic profiling of mTOR-related epilepsy

Malformations of cortical development (MCDs) are a major cause of drug-resistant epilepsy (DRE) in children. A subset of MCDs, including focal cortical dysplasia type II, tuberous sclerosis complex, and hemimegalencephaly, shares characteristic histopathological features and somatic mutations in genes within the PI3K-AKT-mTOR signaling cascade, and is termed mTORopathies. However, the molecular mechanisms that drive epileptogenesis and consequent uncontrollable seizures in these disorders remain poorly understood. Here, we performed integrated genomic, transcriptomic, and proteomic analyses of 60 surgical brain specimens from 42 patients with MCDs and 18 with mesial temporal lobe epilepsy. Targeted genomic sequencing identified 19 somatic mutations in PI3K-AKT-mTOR pathway genes, including novel variants in AKT3 (L51H) and MTOR (A512T). RNA sequencing comparing mTORopathy brains with controls, along with cross-validation using an independent public dataset, identified 287 consistently differentially expressed genes that were consistently altered. Downregulated genes were enriched in oxidative phosphorylation (OXPHOS) and mitochondrial metabolic pathways, whereas upregulated genes were associated with gliogenesis and cellular senescence. Proteomic profiling identified 44 differentially expressed proteins, including significant reductions in OXPHOS-related proteins (COX5B, NDUFS4, GOT2, RAB27B, and DKK3). Our study provides a patient-matched, multi-omics dataset of human mTOR-related epilepsies from a single-institution cohort. Integrated analyses across transcriptomic and proteomic layers highlight impairment of OXPHOS as a molecular hallmark of mTORopathies, Integrated transcriptomic and proteomic analyses identified impaired OXPHOS as a molecular hallmark, potentially contributing to focal hypometabolism in mTORopathy. These findings offer mechanistic insights into epileptogenesis and provide a valuable resource for understanding molecular pathologies and developing mechanism-based therapies for mTOR-related epilepsy.

neuroscience↗

bioRxiv2026

Intra-annual precipitation variability mutes or magnifies the impact of wet and dry years on plant biomass.

As the atmosphere warms, both the amount and timing of precipitation are changing, but how those two trends interact to influence plant growth remains unresolved. Using 5-15 years of data from 48 globally distributed grassland sites, we quantified how the temporal distribution of precipitation within the year (evenness) interacts with annual precipitation amount and nutrient limitation to influence plant biomass. Annual precipitation anomalies had a large influence on plant biomass when intra-annual precipitation was more evenly distributed (frequent small events) and little impact when less even (infrequent large events). This relationship was consistent across aridity gradients and nutrient limitations and strongest in systems with warm wet seasons. Our work shows that the response of plant systems to changes in annual precipitation amount are largely dependent on how evenly it is temporally distributed.

ecology↗

bioRxiv2026

Single-cell-resolved spatial multi-omics identifies mTOR-driven neuronal and astroglial pathogenicity underlying epileptogenic focal cortical dysplasia

Focal cortical dysplasia type II (FCDII) is a malformation of cortical development caused by somatic mutations in the mTOR signaling pathway. Two hallmark pathological cell types in FCDII, dysmorphic neurons (DNs) and balloon cells (BCs), arise as a result of somatic mutations in the mTOR signaling pathway and are implicated in the pathophysiology of drug-resistant epilepsy. However, how these somatic mutations reshape cell states within the human cortex remains poorly understood. Here, we integrate imaging-based spatial transcriptomics (iST), single-nucleus RNA sequencing, and proteomics of surgically resected FCDIIb tissue to define the transcriptional and proteomic profiles of DNs and BCs. Spatial mapping of iST data resolved transcriptional signatures in histologically validated DNs and BCs within FCDIIb sections. Integrative omics analysis further revealed that DNs show upregulation of PI3K-AKT-mTOR and p53-CROT metabolic programs accompanied by suppression of synaptic signaling, whereas BCs exhibit transcriptional signatures of reactive astrocytes with increased phagocytic and immune-like activity. These data delineate cell-type-specific consequences of somatic mTOR pathway mutations at single-cell resolution and reveal previously unrecognized metabolic and immunoregulatory mechanisms contributing to epileptogenesis in drug-resistant epilepsy. Our study establishes a spatial multi-omics framework for dissecting human cortical malformations and highlights potential therapeutic targets for drug-resistant epilepsy.

neuroscience↗

bioRxiv2025

Pseudovirus-Mediated Proximity Labeling Identifies Candidate Host Cell Membrane Proteins Involved in Viral Attachment

Viral infections represent a significant threat to humanity, as exemplified by the COVID-19 pandemic. To mitigate the associated damage, it is essential to understand the biological characteristics of causative viruses. In this study, we report a novel method for identifying host cell molecules (including virus receptor) required for viral attachment, referred to here as "Host Cell Attachment Factors (HCAFs)". Our approach utilizes proximity labeling (PL) technology: pseudoviruses engineered to express a PL enzyme are applied to host cells, where they bind and initiate proximity labeling. Since HCAFs are expected to be localized near viral attachment sites, candidate HCAFs are specifically tagged by the PL process. Analysis of influenza HCAFs enabled the identification of multiple candidate molecules. Subsequent virus attachment experiments using CHO cells suggested that NRP1 may serve as an HCAF for influenza viruses. Given its simplicity and rapid turnaround, this method is adaptable to a wide range of enveloped viruses, including pandemic viruses that require emergency analysis.

microbiology↗

bioRxiv2024

Novel endornaviruses infecting Phytophthora cactorum that attenuate vegetative growth, promote sporangia formation, and confer hypervirulence to the host oomycete.

Two novel endornaviruses were found in Phytophthora cactorum isolated from severe black lesions on Boehmeria nivea (B. nivea) var. nipononivea plants in a Japanese forest. These two endornaviruses were named Phytophthora cactorum alphaendornavirus 4 (PcAEV4) and Phytophthora cactorum alphaendornavirus 5 (PcAEV5), and have site-specific nick structures in their positive RNA strands, which are hallmarks of alphaendornaviruses. Ribavirin and cycloheximide treatment of the protoplasts effectively cured the host oomycete (Kara1) of the viruses. The resultant virus-free strain (Kara1-C) displayed luxuriant mycelial growth with less zoosporangia formation as compared to the Kara1 strain. Remarkably, the Kara1-C strain exhibited reduced ability to form black lesions on B. nivea leaves, suggesting that presence of PcAEV4 and PcAEV5 in the Kara1 strain led to enhanced virulence in host plants. Under osmotic pressure and cell wall synthesis inhibition, the Kara1 strain exhibited less growth inhibition compared with the Kara1-C strain. In contrast, the Kara1 strain showed more growth inhibition to membrane-permeable surfactant compared with the Kara1-C strain, indicating that these endornaviruses can alter the susceptibility of the host oomycete to abiotic stresses. Co-localization and cell fractionation analyses showed that PcAEV4 and PcAEV5 localized to intracellular membranes, particularly the ER membrane fraction. Further, infection with these 2 endornaviruses was found to affect the hosts response to exogenous sterols, which enhanced vegetative growth and zoosporangia formation, as well as virulence of the host oomycete. These results provide deep insights into the effects of endornavirus infection in the Phytophthora spp. and also highlight protoplast-based methods in advancing Phytophthora virus studies.

microbiology↗

bioRxiv2024

Identification of a novel mycovirus belonging to the ''flexivirus''-related family with icosahedral virion

The order Tymovirales currently comprises five viral families with positive-sense RNA ((+)RNA) genomes that infect plants, fungi, and insects. Virion morphologies within the order Tymovirales differ between families, with icosahedral virions in the Tymoviridae and filamentous virions in the other "flexi"viridae families. Despite their different morphologies, these viruses are placed in the same order based on phylogenetic analyses of replicase-associated polyproteins. However, one of the families in the Tymovirales, Deltaflexiviridae, is considered to be capsidless because there have been no published reports of virion isolation. Here we report that a new "flexivirus"-related (+)RNA virus, prospectively named Fusarium oxysporum icosahedral virus 1 (FoIV1), is icosahedral, and that most deltaflexiviruses may have icosahedral virions. Phylogenetic analyses based on replicase-associated polyproteins indicated that FoIV1 forms a distinct group in the Tymovirales with some viruses originally assigned to the Deltaflexiviridae. Electron microscopy, protein analysis, and protein structure predictions indicate that FoIV1 ORF4 encodes a single jelly-roll (SJR)-like coat protein (CP) that constitutes the icosahedral virions. Results of clustering analyses based on amino acid sequences and predicted CP structures suggested that most of the deltaflexiviruses have icosahedral virions composed of SJR-like CPs as in FoIV1, rather than having filamentous virions or capsidless. These results challenge the conventional understanding of viruses in the order Tymovirales, with important implications for revising its taxonomic framework and providing insights into the evolutionary relationships within this diverse and broad host range group of (+)RNA viruses.

microbiology↗

bioRxiv2023

LsTT2 encoding R2R3-MYB transcription factor is responsible for a shift from black to white in lettuce seed

Prickly lettuce (Lactuca serriola), which is considered the wild ancestor of lettuce, has black seeds, whereas the major seed color of domesticated lettuce is black or white. The successfully-selected white seed trait is a key domestication trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black to white seeds remains to be clarified. We aimed to identify the gene/s responsible for white seed trait in lettuce. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F2 population derived from a cross between ShinanoPower (white seed) and Escort (black seed). The white seed trait was controlled by a single recessive locus (48.055-50.197 Mbp) in linkage group 7. Narrowing down using five PCR-based markers and 84 cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49.251Mbp_HinfI marker, which employs a single nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020.1 was completely linked to the seed color phenotype. In addition, the sequences of the coding region for candidate genes except for Lsat_1_v5_gn_7_35020.1 were identical in the resequence analysis of ShinanoPower (white seed) and Escort (black seed). Therefore, we proposed Lsat_1_v5_gn_7_35020.1, a gene located in the locus, as the candidate gene and designated it as LsTT2, an ortholog encoding the R2R3 MYB transcription factor in Arabidopsis. When we validated the role of LsTT2 in seed color through genome editing, LsTT2 knockout mutants harboring an early termination codon showed a change in seed color from black to white. White seeds accumulated less proanthocyanidins than black seeds, which was similar to the phenotype observed in Arabidopsis TRANSPARENT TESTA 2 (TT2) mutants. Therefore, LsTT2 was the allele responsible for the shift in seed color from black to white. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds has implications for future breeding technology and physiological analysis.

genetics↗

bioRxiv2022

Characterization and visualization of global metabolomic responses of Brachypodium distachyon to environmental changes

Plant responses to environmental change are mediated via changes in cellular metabolomes. However, <5% of signals obtained from tandem liquid chromatography mass spectrometry (LC-MS/MS) can be identified, limiting our understanding of how different metabolite classes change under biotic/abiotic stress. To address this challenge, we performed untargeted LC-MS/MS of leaves, roots and other organs of Brachypodium distachyon, a model Poaceae species, under 17 different organ-condition combinations, including copper deficiency, heat stress, low phosphate and arbuscular mycorrhizal symbiosis (AMS). We used a combination of information theory-based metrics and machine learning-based identification of metabolite structural classes to assess metabolomic changes. Both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We also found that one week of copper deficiency shielded the root metabolome, but not the leaf metabolome, from perturbation due to heat stress. Using a recently published deep learning based method for metabolite class predictions, we analyzed the responsiveness of each metabolite class to environmental change, which revealed significant perturbations of various lipid classes and phenylpropanoids such as cinnamic acids and flavonoids. Co-accumulation analysis further identified condition-specific metabolic biomarkers. Finally, to make these results publicly accessible, we developed a novel visualization platform on the Bioanalytical Resource website, where significantly perturbed metabolic classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and plant stress adaptation.

plant biology↗

bioRxiv2021

Size-dependent susceptibility of lake phytoplankton to light stress: An implication for succession of large green algae in a deep oligotrophic lake

Field observations of the population dynamics and measurements of photophysiology in Lake Biwa were conducted by size class (< vs. > 30 m) from early summer to autumn to investigate the relationships between susceptibility to light stress and cell size. Also, a nutrient bioassay was conducted to clarify whether the growth rate and photosystem II (PSII) photochemistry of small and large phytoplankton are limited by nutrient availability. Large phytoplankton, which have lower intracellular Chl-a concentrations, had higher maximum PSII photochemical efficiency (Fv/Fm) but lower non-photochemical quenching (NPQNSV) than small phytoplankton under both dark and increased light conditions. The nutrient bioassay revealed that the PSII photochemistry of small phytoplankton was restricted by N and P deficiency at the pelagic site even at the end of the stratification period, while that of large phytoplankton was not. These results suggest that large phytoplankton have lower susceptibility to PSII photodamage than small phytoplankton due to lower intracellular Chl-a concentrations. The size dependency of susceptibility to PSII photoinactivation may play a key role in large algal blooms in oligotrophic water.

microbiology↗

bioRxiv2021

Seroepidemiological survey on pigs and cattle for novel K88 (F4)-like colonization factor detected in human enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that express various fimbrial or nonfimbrial colonization factors and enterotoxins are critical causes of diarrheal diseases. Human ETEC serotype O169:H41 (O169) has been a representative of epidemic ETEC worldwide; the organism shows massive adherence to HEp-2 cells similar to enteroaggregative E. coli. Previously, we determined the complete sequence of the unstable virulence plasmid, pEntYN10. The plasmid included a unique set of genes encoding a novel colonization factor (CF) resembling K88 (F4) of porcine ETEC, in addition to CS6, a well-known representative CF of human ETEC, and another novel CF similar to CS8 (CFA/III) of human ETEC. To determine whether the K88-like CF (after this, K88O169) allows the organisms to infect domestic animals like the original K88-harboring strains that can cause diarrhea in piglets, samples were tested for antibodies against recombinant proteins of possible paralogous adhesins, FaeG1 and FaeG2, from K88O169 and the FaeG of typical K88 (F4). The seroepidemiological study using recombinant antigens (two paralogs FaeG1 and FaeG2 from K88O169) showed reactivity of porcine (18.0%) and bovine (17.1%) sera to K88O169 FaeG1 and/or FaeG2 antigens on indirect ELISA tests. These results suggest that E. coli with K88O169 adhesin can infect various hosts, including pigs and cattle. This is the first report of domestic animals having antibodies to K88O169 of human ETEC. Although human ETEC had been thought to be distinguished from those of domestic animals based on colonization factors, zoonotic strains may conceal themselves among human ETEC organisms. The concept of One Health should be adopted to intervene in ETEC infections among animals and humans.

microbiology↗

bioRxiv2020

A pair of effectors encoded on a conditionally dispensable chromosome of Fusarium oxysporum suppress host-specific immunity

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute significantly to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis and cabbage. Here we show that Focn has multiple CD chromosomes involved in virulence but also in vegetative growth, which is an atypical feature of CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.

microbiology↗

bioRxiv2020

Development of photosynthetic carbon fixation model using multi-excitation wavelength fast repetition rate fluorometry in Lake Biwa

Direct measurements of gross primary productivity (GPP) in the water column are essential, but can be spatially and temporally restrictive. Fast repetition rate fluorometry (FRRf) is a bio-optical technique based on chlorophyll a (Chl-a) fluorescence that can estimate the electron transport rate (ETRPSII) at photosystem II (PSII) of phytoplankton in real time. However, derivation of phytoplankton GPP in carbon units from ETRPSII remains challenging because the electron requirement for carbon fixation ([FE]e,C) can vary depending on multiple factors. Also, the FRRf is still relatively novel, especially in freshwater ecosystems where phosphorus limitation and cyanobacterial blooms are common. The goal of the present study is to construct a robust [FE]e,C model for freshwater ecosystems using simultaneous measurements of ETRPSII by FRRf with multi-excitation wavelengths coupled with traditional carbon fixation rate by the 13C method. The study was conducted in oligotrophic and mesotrophic areas in Lake Biwa from July 2018 to May 2019. The combination of excitation light at 444, 512 and 633 nm correctly estimated ETRPSII of cyanobacteria. The range of [FE]e,C in the phytoplankton community varied from 1.1 to 31.0 mol e- mol C-1 during the study period. Generalized liner model showed the best model including 12 physicochemical and biological factors explained 67% of the variance in [FE]e,C. Among all factors, water temperature was the most significant, while PAR intensity was not. The GPP values estimated by FRRf (GPPf) with the best [FE]e,C model relative to 13C (GPP13C) varied 0.5-1.5. Further, GPPf estimated with more parsimonious [FE]e,C models were also comparable to GPP13C. This study quantifies the applicability of the in situ FRRf methodology, and supports continuous monitoring of GPP by FRRf in lakes with large spatio-temporal variability of environmental conditions and phytoplankton assemblages.

microbiology↗

bioRxiv2020

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for AMPA-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a new method for the rapid and selective labeling of chemical probes to AMPARs by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological conditions. This method allowed us to quantify AMPAR distribution and trafficking, which revealed some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method was also successfully expanded to selectively label NMDA-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

biochemistry↗

bioRxiv2019

Deglycosylation of isoflavone C-glucoside puerarin by combination of two recombinant bacterial enzymes and 3-oxo-glucose.

C-Glucosides are resistant to glycoside hydrolase activity because the anomeric carbon of glucose is directly connected to aglycone via carbon-carbon bonding. A human intestinal bacterium strain PUE related to Dorea species can metabolize the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) to daidzein and glucose by more than three bacterial enzymes which have not been well-characterized. We previously reported that 3\"-oxo-puerarin is an essential reaction intermediate in enzymatic puerarin degradation and characterized a bacterial enzyme of DgpB-C complex which cleaved the C-glycosidic bond in 3\"-oxo-puerarin. However, the exact enzyme catalyzing the oxidation of C-3\" hydroxyl in puerarin has not been identified, and the other metabolite corresponding to the precursor of D-glucose, derived from the sugar moiety in 3\"-oxo-puerarin in the cleaving reaction catalyzed by the DgpB-C complex, remains unknown.\n\nIn this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3-oxo-glucose as the hydride accepter. In addition, enzymatic C-deglycosylation of puerarin was achieved by a combination of recombinant DgpA, DgpB-C complex, and 3-oxo-glucose. Furthermore, the metabolite derived from the sugar moiety in 3\"-oxo-puerarin cleaving reaction catalyzed by DgpB-C complex was characterized as 1,5-anhydro-D-erythro -hex-1-en-3-ulose, suggesting that the C-glycosidic linkage is cleaved through a {beta}-elimination like mechanism.\n\nImportanceOne important role of the gut microbiota is to metabolize dietary nutrients and supplements such as flavonoid glycosides. Ingested glycosides are metabolized by intestinal bacteria to more absorbable aglycones and further degradation products which show beneficial effects in humans. Although numerous glycoside hydrolases that catalyze O-deglycosylation have been reported, enzymes responsible for C-deglycosylation are still limited. In this study, we characterized enzymes involved in C-deglycosylation of puerarin from a human intestinal bacterium PUE. To our knowledge, this is the first report of the expression, purification and characterization of an oxidoreductase involved in C-glucoside degradation. This study provides new insights for the elucidation of mechanisms of enzymatic C-deglycosylation.

biochemistry↗