Biology subjects

Fushinobu, S.

Publications and source records attributed to Fushinobu, S..

15 recordsLinked to original sources

bioRxiv2026

Crystal structure of a class II UDP-glucose-hexose-1-phosphate uridylyltransferase from Bifidobacterium longum involved in human milk oligosaccharide metabolism

Infant-associated bifidobacteria utilize lacto-N-biose I (LNB) and galacto-N-biose (GNB), major components of human milk oligosaccharides and intestinal mucin, respectively, through the GNB/LNB metabolic pathway. A UDP-glucose-hexose-1-phosphate uridylyltransferase (GalT) from Bifidobacterium longum JCM 1217 (BlGalT2) catalyzes a key step in this pathway. BlGalT2 belongs to class II GalTs within the histidine triad (HIT) superfamily, a rare protein group distinct from canonical class I GalTs. Here, we determined the crystal structure of BlGalT2 at 2.25 [A] resolution, providing the first three-dimensional structure of a class II GalTs. BlGalT2 is a monomeric enzyme with an architecture distinct from canonical class I GalT, comprising characteristic HIT1 and HIT2 catalytic core subdomains and an extended auxiliary domain that forms a self-contained active site. Kinetic analysis revealed a strong preference for GalNAc-1P over Gal-1P, with a 50-fold lower Km for GalNAc-1P, consistent with a hydrophobic pocket accommodating the N-acetyl group. Metal analysis and activity measurements indicated that Zn2+ is required for full activity, and structural prediction suggested its coordination by residues including the first histidine of the HIT motif. AlphaFold3 prediction and mutational analysis identified residues involved in nucleotide-sugar recognition and supported a Ping-Pong reaction mechanism involving a covalent enzyme-uridine monophosphate intermediate. These findings reveal how class II GalTs have evolved a unique structural framework and substrate specificity for N-acetylated sugars. Moreover, the structure of BlGalT2 fills the final missing gap in bifidobacterial HMO metabolism and provides molecular insight into the adaptation of bifidobacteria to host-derived glycans.

biochemistry↗

bioRxiv2026

Structural mechanism defining product specificity in glycoside hydrolase family 66 cycloisomaltotetraose glucanotransferase

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

biochemistry↗

bioRxiv2026

Reprogramming protein interfaces between adenylation and carrier protein domains in nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) assemble structurally diverse bioactive natural products through selective communication between adenylation (A) and carrier protein (CP) domains. Although rational rewiring of these protein-protein interactions could enable customized NRPS design, this remains challenging due to the dynamic nature of protein interfaces. Here we show that structure-guided interface reprogramming enables productive non-cognate A- CP pairings across enterobactin, vibriobactin, pyochelin, and vicenistatin biosynthetic systems. Engineered interactions between the A domains EntE, VibE, or PchD and the non-cognate CPs VinL or EntB were validated by biochemical, kinetic, and structural analyses. Reconstituted pathways containing engineered VibE, EntB, and EntF restored enterobactin production and increased yield to 2.2-fold that of the native EntE-EntB-EntF system. Interface-engineered PchD also enhanced production of a non-native salicylic acid-norspermidine conjugate. An X-ray structure and molecular dynamics simulations of an engineered non-cognate A-CP complex reveal recognition principles for programmable NRPS interface design.

biochemistry↗

bioRxiv2026

Discovery and structural analysis of glycoside hydrolase family 176 α-1,2 glucosidase from Arthrobacter humicola A8F5

Glycoside hydrolases (GHs) exhibit remarkable specificity dictated by the structural configuration of their target glycosidic linkages. While enzymes that process -1,4- and -1,6-linkages in starch or glycogen are well-characterized, those acting on less common bonds, such as -1,2-glucosidic linkages, remain largely underexplored. In this study, we report the discovery and structural elucidation of a novel -1,2-glucosidase from Arthrobacter humicola A8F5 (A8F5 glucosidase), representing a newly uncovered activity within the poorly characterized GH176 family. Biochemical characterizations revealed that A8F5 glucosidase exclusively cleaves -1,2-linkages via an anomer-inverting mechanism, with a distinct preference for short kojioligosaccharides. To circumvent crystallization obstacles caused by high loop flexibility and translational non-crystallographic symmetry, we engineered a loop-truncated variant. This strategy enabled the determination of high-resolution (up to 1.79 [A]) crystal structures of the enzyme in its ligand-free form and in complex with kojibiose, kojitriose, and selaginose. A8F5 glucosidase adopts a (/)6-barrel fold characteristic of clan GH-G. Complementing the crystal structures with AlphaFold3 prediction demonstrated that two prominent active-site loops (loops 3 and 4) adopt a closed conformation that constricts the catalytic pocket, rendering the architecture suitable for short oligosaccharide recognition while restricting access to larger polymers. Furthermore, sequence similarity network analysis highlights vast, uncharacterized functional diversity within the GH176 family. These findings revealed that the GH176 enzyme recognizes and hydrolyses -1,2-glucosidic bonds through a structural framework distinct from that of the previously known clan GH-L GH65 kojibiose hydrolase, expanding the known functional landscape of this enzyme group toward rare -glucans.

biochemistry↗

bioRxiv2025

Neutron crystallography of the covalent intermediate of β-glucosidase reveals remodeling of the catalytic center

Anomer-retaining glycoside hydrolases (GHs) generally catalyze a double displacement reaction via a covalent intermediate; however, neutron crystallography of glycoside ligand-bound states has not been performed. In this study, we investigated {beta}-glucosidase Td2F2 from GH family 1 as a model enzyme for anomer-retaining GHs. We determined joint X-ray/neutron structures of Td2F2 in ligand-free form, covalent intermediate with a 2-deoxy-2-fluoro glucoside (2F-Glc) inhibitor, and glucose product complex using hydrogen/deuterium-exchanged crystals at room temperature, with neutron diffraction resolutions of 1.80-1.70 [A]. Extensive hydrogen bonds recognizing the hydroxy groups of 2F-Glc were identified, along with the positions of deuterium atoms. The acid/base catalyst residue Glu166 was anchored by a hydrogen bond network pivoted by Asn293. Tyr295 forms a hydrogen bond with the catalytic nucleophile residue Glu352 in the ligand-free and glucose complex forms, while the active center undergoes significant reorganization, including side chain displacements of Glu352 and Tyr295, as well as the incorporation of a water molecule. An alternative conformation of Tyr295 was observed in the 2F-Glc structure at room temperature, suggesting its role in positioning the nucleophilic water during the deglycosylation step. The tyrosine hydrogen bonded to the nucleophile is also conserved in many other anomer-retaining GH families, underscoring its importance in catalysis. Based on the deuterium/hydrogen positions determined from neutron structures, we proposed a detailed reaction mechanism for Td2F2. Significance StatementGlycoside hydrolases perform diverse functions in organisms, with over 180 known enzyme families. Although hydrogen bonds and proton transfer play important roles in enzymatic reactions, hydrogen atoms are generally invisible in macromolecular X-ray crystallography. In anomer-retaining glycoside hydrolases, general acid/base catalysis and the formation and hydrolysis of a covalent glycosyl-enzyme intermediate have been postulated. Here, we report neutron crystal structures of a {beta}-glucosidase, where hydrogen and deuterium atoms were visualized at high resolution. An intricate hydrogen-bonding network and remarkable remodeling at the catalytic center were observed during covalent intermediate formation, revealing a detailed catalytic mechanism. The enzyme belongs to glycoside hydrolase family 1 and represents numerous enzymes employing the anomer-retaining mechanism.

biochemistry↗

bioRxiv2024

Crystal structure of plant γ-glutamyl peptidase 1 with dual roles in sulfur metabolism and implications for oxidative stress regulation

{gamma}-Glutamyl peptidase 1 (GGP1) plays a dual role in primary and secondary sulfur metabolism in Arabidopsis thaliana. During glutathione (GSH) turnover, GGP1 hydrolyzes the isopeptide bond of GSH to degrade the tripeptide into Glu and Cys-Gly. During glucosinolate and camalexin biosynthesis, GGP1 processes GSH conjugates, which have a large substituent at the thiol side chain, by hydrolyzing the same isopeptide bond of {gamma}-Glu. In the present study, we determined the crystal structures of the following GGP1 forms: ligand-free, Glu complex, covalent {gamma}-Glu intermediate, and disulfide-linked S-S inactive forms. The intermediate structure, in which {gamma}-Glu is covalently linked to the nucleophile C100, was trapped by mutating the catalytic His to Asn (H192N). In the Glu complex and {gamma}-Glu intermediate structures, Glu bound to the S1 subsite is extensively recognized by several hydrogen bonds. The substrate recognition of the Cys-Gly moiety at the S1 and S2 subsites was revealed by modeling GSH in the active site. Mutational analysis indicated that R206 plays an important role in substrate binding by forming a salt bridge with Gly at the S2 subsite. An open pocket is present beyond the thiol side chain of Cys in the S1 subsite, which contributed to the dual activity of GGP1 toward GSH and GSH conjugates. The S-S inactive structure was obtained by soaking GGP1 crystals in Cys-Gly, and the catalytic cysteine (C100) partially formed a disulfide bond with a neighboring C154 residue. The partial inactivation of GGP1 in the presence of a pro-oxidant (Cys-Gly) has revealed its possible role in oxidative stress regulation in Arabidopsis.

biochemistry↗

bioRxiv2024

Identification and structural characterization of a novel acetyl xylan esterase from Aspergillus oryzae

Acetyl xylan esterase plays a crucial role in the degradation of xylan, the major plant hemicellulose, by liberating acetic acid from the backbone polysaccharides. Acetyl xylan esterase B from Aspergillus oryzae, designated AoAXEB, was biochemically and structurally investigated. The AoAXEB-encoding gene with a native signal peptide was successfully expressed in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 8.0 and 30 {degrees}C, respectively, and was stable up to 35{degrees}C. The optimal substrate for hydrolysis by purified recombinant AoAXEB among a panel of -naphthyl esters was -naphthyl acetate. Recombinant AoAXEB catalyzes the release of acetic acid from wheat arabinoxylan. The release of acetic acid from wheat arabinoxylan increases synergistically with xylanase addition. No activity was detected using the methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. The crystal structures of AoAXEB in the apo and succinate complexes were determined at resolutions of 1.75 and 1.90 [A], respectively. Although AoAXEB has been classified in the Esterase_phb family in the ESTerases and alpha/beta-Hydrolase Enzymes and Relatives (ESTHER) database, its structural features partly resemble those of ferulic acid esterase in the FaeC family. Phylogenetic analysis also indicated that AoAXEB is located between the clades of the two families. Docking analysis provided a plausible binding mode for xylotriose substrates acetylated at the 2- or 3-hydroxy position. This study expands the repertoire of side chain-degrading enzymes required for complete plant biomass degradation.

biochemistry↗

bioRxiv2024

Structural basis for the strict substrate specificity of beta-D-galactofuranosidase from Streptomyces sp. JHA19

D-Galactofuranose (Galf) is widely distributed in polysaccharides and glycoconjugates of bacteria, filamentous fungi, and protozoa. The biosynthetic and degradation pathways of Galf in pathogens have attracted attention as potential targets for drug development. {beta}-D-Galactofuranosidase (Galf-ase) releases Galf from the non-reducing ends of glycans. Galf-ase activity is often exhibited by -L-arabinofuranosidases, which hydrolyze a similar substrate. Several Galf-specific Galf-ases that cleave only Galf and not L-arabinofuranose (Araf) have recently been identified in the glycoside hydrolase (GH) families 2, 5, and 43. However, the structural basis of how they discriminate the substrates is unknown. ORF1110, belonging to GH2, is the first identified Galf-specific Galf-ase isolated from Streptomyces sp. JHA19. Here, we solved the crystal structure of ORF1110 in complex with a mechanism-based potent inhibitor, D-iminogalactitol (Ki = 65 M). ORF1110 binds to the C5-C6 hydroxy groups of D-iminogalactitol with an extensive and integral hydrogen bond network. This result suggests that in the case of Araf, which lacks the C6 hydroxymethyl group, this network is not formed. The domain structure of ORF1110 is similar to that of {beta}-glucuronidases and {beta}-galactosidases, which belong to the same GH2 family and hydrolyze pyranose substrates. However, their active site structures were completely different. A predicted structure of the C-terminal Abf domain of ORF1110 was very similar to the carbohydrate-binding module family 42, which binds Araf, and pockets that may bind Galf were present.

biochemistry↗

bioRxiv2024

Crystal structure of Bifidobacterium bifidum glycoside hydrolase family 110 α-galactosidase specific for blood group B antigen

To overcome incompatibility issues and increase the possibility of blood transfusion, technologies that enable efficient conversion of A- and B-type red blood cells to the universal donor O-type is desirable. Although several blood type-converting enzymes have been identified, detailed understanding about their molecular functions is limited. -Galactosidase from Bifidobacterium bifidum JCM 1254 (AgaBb), belonging to glycoside hydrolase (GH) 110 subfamily A, specifically acts on blood group B antigen. Here we present the crystal structure of AgaBb, including the catalytic GH110 domain and part of the C-terminal uncharacterized regions. Based on this structure, we deduced a possible binding mechanism of blood group B antigen to the active site. Site-directed mutagenesis confirmed that R270 and E380 recognize the fucose moiety in the B antigen. Thermal shift assay revealed that the C-terminal uncharacterized region significantly contributes to protein stability. This region is shared only among GH110 enzymes from B. bifidum and some Ruminococcus species. The elucidation of the molecular basis for the specific recognition of blood group B antigen is expected to lead to the practical application of blood group conversion enzymes in the future.

biochemistry↗

bioRxiv2023

Genetic and functional diversity of beta-N-acetylgalactosamine residue-targeting glycosidases expanded by deep-sea metagenome

{beta}-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. {beta}-N-Acetylgalactosaminidases hydrolyze {beta}-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-{beta}-N-acetylgalactosaminidases that specifically act on {beta}-N-acetylgalactosamine residues, has been documented so far. In this study, we identified three novel glycoside hydrolase families distributed among all three domains of life and characterized eight novel {beta}-N-acetylgalactosaminidases and {beta}-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and diversify their substrate specificities (endo-, dual-endo/exo-, and exo-) through the accumulation of mutations and insertional amino acid sequences. The diverse {beta}-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present novel evolutionary pathways for expanding their substrate specificity.

biochemistry↗

bioRxiv2022

Charge neutralization and β-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography

Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating, and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable tools for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose--1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature up to 1.80 and 1.25 [A] resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuteronated His105 and deuteronated Tyr150 supported this interaction. The unusually hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via {beta}-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them, and this His-His-Asp structural motif is conserved in the three PL families. Significance StatementAlthough hydrogen transfer plays an important role in enzymatic reactions, hydrogen atoms are generally invisible in macromolecular X-ray crystallography. In the reaction of polysaccharide lyases, substrate activation by negative charge stabilization of uronic acid and base/acid-catalyzed {beta}-elimination reaction have been postulated. Here, we report the neutron crystallography of polysaccharide lyase. Joint X-ray/neutron crystallography of L-rhamnose--1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1) complexed with L-rhamnose was performed, and the hydrogen and deuterium atoms were visualized at a high resolution. FoRham1 catalyzes the specific cleavage of the cap structure of gum arabic, which is useful for various applications in the food, cosmetic, and pharmaceutical industries. A detailed catalytic mechanism for FoRham1 was proposed based on the key structural features of its active site.

biophysics↗

bioRxiv2020

Crystallographic and cryogenic electron microscopic structures and enzymatic characterization of sulfur oxygenase reductase from Sulfurisphaera tokodaii

Sulfur oxygenase reductases (SORs) are present in thermophilic and mesophilic archaea and bacteria, and catalyze oxygen-dependent oxygenation and disproportionation of elemental sulfur. SOR has a hollow, spherical homo-24-mer structure and reactions take place at active sites inside the chamber. The crystal structures of SORs from two Acidianus species have been reported. However, the states of the active site components (mononuclear iron and cysteines) and the entry and exit paths of the substrate and products are still in dispute. Here, we report the biochemical and structural characterizations of SORs from the thermoacidophilic archaeon Sulfurisphaera tokodaii (StSOR) and present high-resolution structures determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM). The crystal structure of StSOR was determined at 1.73 [A] resolution. At the catalytic center, iron is ligated to His86, His90, Glu114, and two water molecules. Three conserved cysteines in the cavity are located 9.5[~]13 [A] from the iron and were observed as free thiol forms. A mutational analysis indicated that the iron and one of the cysteines (Cys31) were essential for both activities and the other two cysteines (Cys101 and Cys104) had a supportive role. The cryo-EM structure was determined at 2.24 [A] resolution using an instrument operating at 200 kV. The two structures determined by different methodologies showed similar main chain traces, but the maps exhibited different features at catalytically important components. Given the high resolution achieved in this study, StSOR was shown to be a good benchmark sample for cryo-EM measurements. HighlightsO_LISulfur oxygenase reductase (SOR) was biochemically and structurally characterized. C_LIO_LIHigh resolution structures of SOR were determined by crystallography and cryo-EM. C_LIO_LITwenty-four identical subunits of SOR form a hollow sphere. C_LIO_LICatalytic components exhibited different features in the crystal and cryo-EM structures. C_LI

biochemistry↗

bioRxiv2020

Butyrate producing Clostridiales utilize distinct human milk oligosaccharides correlating to early colonization and prevalence in the human gut

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here we unveil the growth of Roseburia and Eubacterium members on human milk oligosaccharides (HMOs) using an unprecedented catabolic apparatus. The described HMO pathways and additional glycan utilization loci confer co-growth with Akkermansia muciniphilia via cross-feeding and access to mucin O-glycans. Strikingly, both, HMO and xylooligosaccharide pathways, were active simultaneously attesting an adaptation to a mixed HMO-solid food diet. Analyses of 4599 Roseburia genomes underscored the preponderance of HMO pathways and highlighted different HMO utilization phylotypes. Our revelations provide a possible rationale for the early establishment and resilience of butyrate producing Clostridiales and expand the role of specific HMOs in the assembly of the early life microbiota.

microbiology↗

bioRxiv2020

Crystal structure of β-L-arabinobiosidase belonging to glycoside hydrolase family 121

Enzymes acting on -L-arabinofuranosides have been extensively studied; however, the structures and functions of {beta}-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of {beta}-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular {beta}-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing {beta}-1,2-linked arabinofuranose disaccharide ({beta}-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 [A] resolution. The HypBA2 structure consists of a central catalytic (/)6 barrel domain and two flanking (N- and C-terminal) {beta}-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the {beta}-Ara2 disaccharide; this pocket is highly conserved among GH121 proteins. The three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all ~270 members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of GH63 -glycosidase, GH94 chitobiose phosphorylase, GH142 {beta}-L-arabinofuranosidase, GH78 -L-rhamnosidase, and GH37 ,-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. {beta}-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the {beta}-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.

biochemistry↗

bioRxiv2017

Structure Of Allium Lachrymatory Factor Synthase Elucidates Catalysis On Sulfenic Acid Substrate

Natural lachrymatory effects are invoked by small volatile S-oxide compounds. They are produced through alkene sulfenic acids by the action of lachrymatory factor synthase (LFS). Here we present the crystal structures of onion LFS (AcLFS) revealed in solute-free and two solute-stabilized forms. Each structure adopts a single seven-stranded helix-grip fold possessing an internal pocket. Mutagenesis analysis localized the active site to a layer near the bottom of the pocket, which is adjacent to the deduced key residues Arg71, Glu88, and Tyr114. Solute molecules visible on the active site have suggested that AcLFS accepts various small alcohol compounds as well as its natural substrate, and they inhibit this substrate according to their chemistry. Structural homologs have been found in the SRPBCC superfamily, and comparison of the active sites has demonstrated that the electrostatic potential unique to AcLFS could work in capturing the substrate in its specific state. Finally, we propose a rational catalytic mechanism based on intramolecular proton shuttling in which the microenvironment of AcLFS can bypass the canonical [1,4]-sigmatropic rearrangement principle established by microwave studies. Beyond revealing how AcLFS generates the lachrymatory compound, this study provides insights into the molecular machinery dealing with highly labile organosulfur species.\n\nSignificance statementCrushing of onion liberates a volatile compound, syn-propanethial S-oxide (PTSO), which causes lachrymatory effect on humans. We present the crystal structures of onion LFS (AcLFS), the enzyme responsible for natural production of PTSO. AcLFS features a barrel-like fold, and mutagenic and inhibitory analyses revealed that the key residues are present in the central pocket, harboring highly concentrated aromatic residues plus a dyad motif. The architecture of AcLFS is widespread among proteins with various biological functions, such as abscisic acid receptors and polyketide cyclases, and comparisons with these homologs indicate that unique steric and electronic properties maintain the pocket as a reaction compartment. We propose the molecular mechanism behind PTSO generation and shed light on biological decomposition of short-lived sulfur species.

biochemistry↗