Structure-function analysis of bacterial GH31 α-galactosidases specific for α-(1→4)-galactobiose.

Glycoside hydrolase family 31 (GH31) contains α-glycoside hydrolases with different substrate specificities involved in various physiological functions. This family has recently been classified into 20 subfamilies using sequence similarity networks. An α-galactosidase from the gut bacterium Bacteroides salyersiae (BsGH31_19, which belongs to GH31 subfamily 19) was reported to have hydrolytic activity against the synthetic substrate p- nitrophenyl α-galactopyranoside, but its natural substrate remained unknown. BsGH31_19 shares low sequence identity (around 20%) with other reported GH31 α-galactosidases, PsGal31A from Pseudopedobacter saltans and human myogenesis-regulating glycosidase (MYORG), and was expected to have distinct specificity. Here, we characterized BsGH31_19 and its ortholog from a soil Bacteroidota bacterium, Flavihumibacter petaseus (FpGH31_19), and demonstrated that they showed high substrate specificity against α-(1→4)-linkages in α-(1→4)-galactobiose and globotriose [α-Gal-(1→4)-ß-Gal-(1→4)-Glc], unlike PsGal31A and MYORG. The crystallographic analyses of BsGH31_19 and FpGH31_19 showed that their overall structures resemble those of MYORG and form a dimer with an interface different from that of PsGal31A and MYORG dimers. The structures of FpGH31_19 complexed with d-galactose and α-(1→4)-galactobiose revealed that amino acid residues that recognize a galactose residue at subsite +1 are not conserved between FpGH31_19 and BsGH31_19. The tryptophan (Trp153) that recognizes galactose at subsite -1 is homologous to the tryptophan residues in MYORG and α-galactosidases belonging to GH27, GH36, and GH97, but not in the bacterial GH31 member PsGal31A. Our results provide structural insights into molecular diversity and evolutionary relationships in the GH31 α-galactosidase subfamilies and the other α-galactosidase families.

Crystal structure and sugar-binding ability of the C-terminal domain of N-acetylglucosaminyltransferase IV establish a new carbohydrate-binding module family.

N-glycans are modified by glycosyltransferases in the endoplasmic reticulum and Golgi apparatus. N-acetylglucosaminyltransferase IV (GnT-IV) is a Golgi-localized glycosyltransferase that synthesizes complex-type N-glycans in vertebrates. This enzyme attaches N-acetylglucosamine (GlcNAc) to the α-1,3-linked mannose branch of the N-glycan core structure via a ß-1,4 linkage. Deficiency of this enzyme is known to cause abnormal cellular functions, making it a vital enzyme for living organisms. However, there has been no report on its 3-dimensional structure to date. Here, we demonstrated that the C-terminal regions (named CBML) of human GnT-IVa and Bombyx mori ortholog have the ability to bind ß-N-acetylglucosamine. In addition, we determined the crystal structures of human CBML, B. mori CBML, and its complex with ß-GlcNAc at 1.97, 1.47, and 1.15 Å resolutions, respectively, and showed that they adopt a ß-sandwich fold, similar to carbohydrate-binding module family 32 (CBM32) proteins. The regions homologous to CBML (≥24% identity) were found in GnT-IV isozymes, GnT-IVb, and GnT-IVc (known as GnT-VI), and the structure of B. mori CBML in complex with ß-GlcNAc indicated that the GlcNAc-binding residues were highly conserved among these isozymes. These residues are also conserved with the GlcNAc-binding CBM32 domain of ß-N-acetylhexosaminidase NagH from Clostridium perfringens despite the low sequence identity (<20%). Taken together with the phylogenetic analysis, these findings indicate that these CBMLs may be novel CBM family proteins with GlcNAc-binding ability.

Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides.

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The kcat/Km values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.

Structural and mechanistic insights into the substrate specificity and hydrolysis of GH31 α-N-acetylgalactosaminidase.

Glycoside hydrolase family 31 (GH31) is a diversified family of anomer-retaining α-glycoside hydrolases, such as α-glucosidase and α-xylosidase, among others. Recently, GH31 α-N-acetylgalactosaminidases (Nag31s) have been identified to hydrolyze the core of mucin-type O-glycans and the crystal structure of a gut bacterium Enterococcus faecalis Nag31 has been reported. However, the mechanisms of substrate specificity and hydrolysis of Nag31s are not well investigated. Herein, we show that E. faecalis Nag31 has the ability to release N-acetylgalactosamine (GalNAc) from O-glycoproteins, such as fetuin and mucin, but has low activity against Tn antigen. Mutational analysis and crystal structures of the Michaelis complexes reveal that residues of the active site work in concert with their conformational changes to act on only α-N-acetylgalactosaminides. Docking simulations using GalNAc-attached peptides suggest that the enzyme mainly recognizes GalNAc and side chains of Ser/Thr, but not strictly other peptide residues. Moreover, quantum mechanics calculations indicate that the enzyme preferred p-nitrophenyl α-N-acetylgalactosaminide to Tn antigen and that the hydrolysis progresses through a conformational itinerary, 4C1 → 1S3 → 4C1, in GalNAc of substrates. Our results provide novel insights into the diversification of the sugar recognition and hydrolytic mechanisms of GH31 enzymes.

Ferulic acid alleviates abnormal behaviors in isolation-reared mice via 5-HT1A receptor partial agonist activity.

RATIONALE: Preclinical and clinical reports suggest that ferulic acid (FA), a plant-derived phenylpropanoid, is effective against mental health problems such as agitation, anxiety, and irritability in humans, without causing adverse side effects. However, the mechanism of action is unknown. OBJECTIVE: The aim of the study is to investigate the mechanism underlying the ameliorative effects of FA on mental health problems such as agitation, anxiety, and irritability, using in vivo behavioral analysis, in vitro pharmacological analysis, and in silico binding analysis. METHODS: The effects of FA (10 mg/kg, 50 mg/kg, and 250 mg/kg) on hyperactivity and aggressive behaviors of isolation-reared mice were examined. The effects of FA (50 mg/kg and 250 mg/kg) on extracellular levels of monoamines such as serotonin (5-HT), dopamine, and noradrenaline were analyzed by in vivo microdialysis. The effects of FA (10-13-10-6 M) on 5-HT1A and 5-HT2A receptors were analyzed using a luciferase reporter gene assay. Binding of FA to the mouse 5-HT1A receptor was evaluated by in silico analysis. RESULTS: The behavioral analysis showed that administration of FA (50 mg/kg) 1 h before experiments significantly alleviated hyperactivity and aggressive behaviors in isolation-reared mice. These alleviative effects were abolished by pretreatment with the 5-HT1A receptor antagonist WAY-100635 (1 mg/kg). In vivo microdialysis analysis showed that FA (50 mg/kg) did not change extracellular monoamine levels in the prefrontal cortex of mice. The luciferase reporter gene assay indicated that FA activated 5-HT1A receptors, but not 5-HT2A receptors, in a dose-dependent manner. The maximal response of 5-HT1A receptors to FA was weaker than that to 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT), a 5-HT1A receptor full agonist. In silico binding analysis showed that FA binds to the orthosteric site of 5-HT1A receptors. CONCLUSION: Taken together, these results suggest that FA ameliorates agitation-, anxiety-, and irritability-like behaviors such as hyperactivity and aggressive behaviors in isolation-reared mice via 5-HT1A receptor partial agonist activity. These findings support the efficacy of FA on mental health problems that have been suggested in preclinical and clinical practice.

Biochemical characterization and mutational analysis of silkworm Bombyx mori ß-1,4-N-acetylgalactosaminyltransferase and insight into the substrate specificity of ß-1,4-galactosyltransferase family enzymes.

Silkworm Bombyx mori is one of the insect hosts for recombinant protein production at academic and industrial levels. B. mori and other insect cells can produce mammalian proteins with proper posttranslational modifications, such as N-glycosylation, but the structures of N-glycans in B. mori are mainly high mannose- and paucimannose-type, while mammals also produce hybrid- and complex-type glycans. Recently, complex-type N-glycans whose structures are different from mammalian ones have been identified in some insect cell N-glycomes at very low levels compared with levels of high mannose- and paucimannose-type glycans. However, their functions and the enzymes involved in the biosynthesis of insect complex-type N-glycans are not clear, and complex-type N-glycans, except for N-acetylglucosamine-terminated glycans, are still not identified in the B. mori N-glycome. Here, we focused on the ß-1,4-galactosyltransferase family (also known as glycosyltransferase family 7, GT7) that contains mammalian ß-1,4-galactosyltransferase and insect ß-1,4-N-acetylgalactosaminyltransferase. A gene for a GT7 protein (BmGalNAcT) from B. mori was cloned, expressed in a soluble form using a silkworm expression system, and the gene product showed strict ß-1,4-N-acetylgalactosaminyltransferase activity but not ß-1,4-galactosyltransferase activity. A mutation in Ile298 or Ile310, which are predicted to be located in the active site, reduced its glycosyltransferase activity, suggesting that these residues and the corresponding residues are responsible for substrate specificity of GT7. These results suggested that BmGalNAcT may be involved in the complex-type N-glycans, and moreover, bioinformatics analysis revealed that B. mori might have an extra gene for a GT7 enzyme with different specificity in addition to the known insect GT7 glycosyltransferases.