Structural basis for the recognition of α-1,6-branched α-glucan by GH13_47 α-amylase from Rhodothermus marinus.

Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides.

Structural insights into α-(1→6)-linkage preference of GH97 glucodextranase from Flavobacterium johnsoniae.

Glycoside hydrolase family 97 (GH97) comprises enzymes like anomer-inverting α-glucoside hydrolases (i.e., glucoamylase) and anomer-retaining α-galactosidases. In a soil bacterium, Flavobacterium johnsoniae, we previously identified a GH97 enzyme (FjGH97A) within the branched dextran utilization locus. It functions as an α-glucoside hydrolase, targeting α-(1→6)-glucosidic linkages in dextran and isomaltooligosaccharides (i.e., glucodextranase). FjGH97A exhibits a preference for α-(1→6)-glucoside linkages over α-(1→4)-linkages, while Bacteroides thetaiotaomicron glucoamylase SusB (with 69% sequence identity), which is involved in the starch utilization system, exhibits the highest specificity for α-(1→4)-glucosidic linkages. Here, we examined the crystal structures of FjGH97A in complexes with glucose, panose, or isomaltotriose, and analyzed the substrate preferences of its mutants to identify the amino acid residues that determine the substrate specificity for α-(1→4)- and α-(1→6)-glucosidic linkages. The overall structure of FjGH97A resembles other GH97 enzymes, with conserved catalytic residues similar to anomer-inverting GH97 enzymes. A comparison of active sites between FjGH97A and SusB revealed differences in amino acid residues at subsites +1 and +2 (specifically Ala195 and Ile378 in FjGH97A). Among the three mutants (A195S, I378F, and A195S-I378F), A195S and A195S-I378F exhibited increased activity toward α-(1→4)-glucoside bonds compared to α-(1→6)-glucoside bonds. This suggests that Ala195, located on the Gly184-Thr203 loop (named loop-N) conserved within the GH97 subgroup, including FjGH97A and SusB, holds significance in determining linkage specificity. The conservation of alanine in the active site of the GH97 enzymes, within the same gene cluster as the putative dextranase, indicates its crucial role in determining the specificity for α-(1→6)-glucoside linkage.

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.

Bacteroidota polysaccharide utilization system for branched dextran exopolysaccharides from lactic acid bacteria.

Dextran is an α-(1→6)-glucan that is synthesized by some lactic acid bacteria, and branched dextran with α-(1→2)-, α-(1→3)-, and α-(1→4)-linkages are often produced. Although many dextranases are known to act on the α-(1→6)-linkage of dextran, few studies have functionally analyzed the proteins involved in degrading branched dextran. The mechanism by which bacteria utilize branched dextran is unknown. Earlier, we identified dextranase (FjDex31A) and kojibiose hydrolase (FjGH65A) in the dextran utilization locus (FjDexUL) of a soil Bacteroidota Flavobacterium johnsoniae and hypothesized that FjDexUL is involved in the degradation of α-(1→2)-branched dextran. In this study, we demonstrate that FjDexUL proteins recognize and degrade α-(1→2)- and α-(1→3)-branched dextrans produced by Leuconostoc citreum S-32 (S-32 α-glucan). The FjDexUL genes were significantly upregulated when S-32 α-glucan was the carbon source compared with α-glucooligosaccharides and α-glucans, such as linear dextran and branched α-glucan from L. citreum S-64. FjDexUL glycoside hydrolases synergistically degraded S-32 α-glucan. The crystal structure of FjGH66 shows that some sugar-binding subsites can accommodate α-(1→2)- and α-(1→3)-branches. The structure of FjGH65A in complex with isomaltose supports that FjGH65A acts on α-(1→2)-glucosyl isomaltooligosaccharides. Furthermore, two cell surface sugar-binding proteins (FjDusD and FjDusE) were characterized, and FjDusD showed an affinity for isomaltooligosaccharides and FjDusE for dextran, including linear and branched dextrans. Collectively, FjDexUL proteins are suggested to be involved in the degradation of α-(1→2)- and α-(1→3)-branched dextrans. Our results will be helpful in understanding the bacterial nutrient requirements and symbiotic relationships between bacteria at the molecular level.

Utilization of dietary mixed-linkage ß-glucans by the Firmicute Blautia producta.

The ß-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixed-linkage glucans [MLG - ß-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Gram-positive bacteria utilize MLG largely remains unknown. In this study, we used Blautia producta ATCC 27340 as a model organism to develop an understanding of MLG utilization. B. producta encodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16MLG), an ABC transporter, and a glycoside phosphorylase (BpGH94MLG) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)-encoding genes in this cluster when the organism is grown on MLG. We determined that recombinant BpGH16MLG cleaved various types of ß-glucan, generating oligosaccharides suitable for cellular uptake by B. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinant BpGH94MLG and ß-glucosidases (BpGH3-AR8MLG and BpGH3-X62MLG). Using targeted deletion, we demonstrated BpSBPMLG is essential for B. producta growth on barley ß-glucan. Furthermore, we revealed that beneficial bacteria, such as Roseburia faecis JCM 17581T, Bifidobacterium pseudocatenulatum JCM 1200T, Bifidobacterium adolescentis JCM 1275T, and Bifidobacterium bifidum JCM 1254, can also utilize oligosaccharides resulting from the action of BpGH16MLG. Disentangling the ß-glucan utilizing the capability of B. producta provides a rational basis on which to consider the probiotic potential of this class of organism.

Identification and Characterization of Dextran α-1,2-Debranching Enzyme from Microbacterium dextranolyticum.

Dextran α-1,2-debranching enzyme (DDE) releases glucose with hydrolyzing α-(1→2)-glucosidic linkages in α-glucans, which are made up of dextran with α-(1→2)-branches and are generated by Leuconostoc bacteria. DDE was isolated from Microbacterium dextranolyticum (formerly known as Flavobacterium sp. M-73) 40 years ago, although the amino acid sequence of the enzyme has not been determined. Herein, we found a gene for this enzyme based on the partial amino acid sequences from native DDE and characterized the recombinant enzyme. DDE had a signal peptide, a glycoside hydrolase family 65 domain, a carbohydrate-binding module family 35 domain, a domain (D-domain) similar to the C-terminal domain of Arthrobacter globiformis glucodextranase, and a transmembrane region at the C-terminus. Recombinant DDE released glucose from α-(1→2)-branched α-glucans produced by Leuconostoc citreum strains B-1299, S-32, and S-64 and showed weak hydrolytic activity with kojibiose and kojitriose. No activity was detected for commercial dextran and Leuconostoc citreum B-1355 α-glucan, which do not contain α-(1→2)-linkages. The removal of the D-domain decreased the affinity for α-(1→2)-branched α-glucans but not for kojioligosaccharides, suggesting that D-domain plays a role in α-glucan binding. Genes for putative dextranases, oligo-1,6-glucosidase, sugar-binding protein, and permease were present in the vicinity of the DDE gene, and as a result these gene products may be necessary for the use of α-(1→2)-branched glucans. Our findings shed new light on how actinobacteria utilize polysaccharides produced by lactic acid bacteria.

Glycoside hydrolases active on microbial exopolysaccharide α-glucans: structures and function.

Glucose is the most abundant monosaccharide in nature and is an important energy source for living organisms. Glucose exists primarily as oligomers or polymers and organisms break it down and consume it. Starch is an important plant-derived α-glucan in the human diet. The enzymes that degrade this α-glucan have been well studied as they are ubiquitous throughout nature. Some bacteria and fungi produce α-glucans with different glucosidic linkages compared with that of starch, and their structures are quite complex and not fully understood. Compared with enzymes that degrade the α-(1→4) and α-(1→6) linkages in starch, biochemical and structural studies of the enzymes that catabolize α-glucans from these microorganisms are limited. This review focuses on glycoside hydrolases that act on microbial exopolysaccharide α-glucans containing α-(1→6), α-(1→3), and α-(1→2) linkages. Recently acquired information regarding microbial genomes has contributed to the discovery of enzymes with new substrate specificities compared with that of previously studied enzymes. The discovery of new microbial α-glucan-hydrolyzing enzymes suggests previously unknown carbohydrate utilization pathways and reveals strategies for microorganisms to obtain energy from external sources. In addition, structural analysis of α-glucan degrading enzymes has revealed their substrate recognition mechanisms and expanded their potential use as tools for understanding complex carbohydrate structures. In this review, the author summarizes the recent progress in the structural biology of microbial α-glucan degrading enzymes, touching on previous studies of microbial α-glucan degrading enzymes.

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.

Unlocking the Hydrolytic Mechanism of GH92 α-1,2-Mannosidases: Computation Inspires the use of C-Glycosides as Michaelis Complex Mimics.

The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via O S2 →B2,5 →1 S5 and 3 S1 →3 H4 →1 C4 . For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4 C1 →4 H5 /1 S5 →1 S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH's natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5 →B2,5 /1 S5 →1 S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.

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.

Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases.

Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu472 was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu616 instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family.

Structural insight into the substrate specificity of Bombyx mori ß-fructofuranosidase belonging to the glycoside hydrolase family 32.

Sucrose-hydrolyzing enzymes are largely divided into ß-fructofuranosidase and sucrose α-glucosidase. The domestic silkworm Bombyx mori possesses both enzymes, BmSUC1 and BmSUH, belonging to the glycoside hydrolase family 32 (GH32) and GH13, respectively. BmSUC1 was presumed to be acquired by horizontal gene transfer from bacteria based on phylogenetic analysis and related to tolerance to sugar-mimic alkaloids contained in mulberry latex. Here we investigated the substrate specificity of recombinant BmSUC1 that can hydrolyze not only sucrose but also fructooligosaccharides and fructans, and revealed that the enzyme was competitively inhibited by 1,4-dideoxy-1,4-imino-D-arabinitol, one of the alkaloids. Moreover, the crystal structures of BmSUC1 in apo form and complex with sucrose were determined, and the active site pocket was shallow and suitable for shorter substrates but was related to more relaxed substrate specificity than the strict sucrose α-glucosidase BmSUH. Considering together with the distribution of BmSUC1-orthologous genes in many lepidopterans, our results suggest that BmSUC1 contributes to the digestion of fructooligosaccharides and fructans derived from feed plants.

α-L-Fucosidase from Bombyx mori has broad substrate specificity and hydrolyzes core fucosylated N-glycans.

N-glycans play a role in physiological functions, including glycoprotein conformation, signal transduction, and antigenicity. Insects display both α-1,6- and α-1,3-linked fucose residues bound to the innermost N-acetylglucosamine of N-glycans whereas core α-1,3-fucosylated N-glycans are not found in mammals. Functions of insect core-fucosylated glycans are not clear, and no α-L-fucosidase related to the N-glycan degradation has been identified. In the genome of the domestic silkworm, Bombyx mori, a gene for a protein, BmFucA, belonging to the glycoside hydrolase family 29 is a candidate for an α-L-fucosidase gene. In this study, BmFucA was cloned and recombinantly expressed as a glutathione-S-transferase tagged protein (GST-BmFucA). Recombinant GST-BmFucA exhibited broad substrate specificity and hydrolyzed p-nitrophenyl α-L-fucopyranoside, 2'-fucosyllactose, 3-fucosyllactose, 3-fucosyl-N,N'-diacetylchitobiose, and 6-fucosyl-N,N'-diacetylchitobiose. Further, GST-BmFucA released fucose from both pyridylaminated complex-type and paucimannose-type glycans that were core-α-1,6-fucosylated. GST-BmFucA also shows hydrolysis activity for core-fucosylated glycans attached to phospholipase A2 from bee venom. BmFucA may be involved in the catabolism of core-fucosylated N-glycans in B. mori.

Structure-function analysis of silkworm sucrose hydrolase uncovers the mechanism of substrate specificity in GH13 subfamily 17 exo-α-glucosidases.

The domestic silkworm Bombyx mori expresses two sucrose-hydrolyzing enzymes, BmSUH and BmSUC1, belonging to glycoside hydrolase family 13 subfamily 17 (GH13_17) and GH32, respectively. BmSUH has little activity on maltooligosaccharides, whereas other insect GH13_17 α-glucosidases are active on sucrose and maltooligosaccharides. Little is currently known about the structural mechanisms and substrate specificity of GH13_17 enzymes. In this study, we examined the crystal structures of BmSUH without ligands; in complexes with substrates, products, and inhibitors; and complexed with its covalent intermediate at 1.60-1.85 Å resolutions. These structures revealed that the conformations of amino acid residues around subsite -1 are notably different at each step of the hydrolytic reaction. Such changes have not been previously reported among GH13 enzymes, including exo- and endo-acting hydrolases, such as α-glucosidases and α-amylases. Amino acid residues at subsite +1 are not conserved in BmSUH and other GH13_17 α-glucosidases, but subsite -1 residues are absolutely conserved. Substitutions in three subsite +1 residues, Gln191, Tyr251, and Glu440, decreased sucrose hydrolysis and increased maltase activity of BmSUH, indicating that these residues are key for determining its substrate specificity. These results provide detailed insights into structure-function relationships in GH13 enzymes and into the molecular evolution of insect GH13_17 α-glucosidases.

Crystal structure of the Enterococcus faecalis α-N-acetylgalactosaminidase, a member of the glycoside hydrolase family 31.

Glycoside hydrolases catalyze the hydrolysis of glycosidic linkages in carbohydrates. The glycoside hydrolase family 31 (GH31) contains α-glucosidase, α-xylosidase, α-galactosidase, and α-transglycosylase. Recent work has expanded the diversity of substrate specificity of GH31 enzymes, and α-N-acetylgalactosaminidases (αGalNAcases) belonging to GH31 have been identified in human gut bacteria. Here, we determined the first crystal structure of a truncated form of GH31 αGalNAcase from the human gut bacterium Enterococcus faecalis. The enzyme has a similar fold to other reported GH31 enzymes and an additional fibronectin type 3-like domain. Additionally, the structure in complex with N-acetylgalactosamine reveals that conformations of the active site residues, including its catalytic nucleophile, change to recognize the ligand. Our structural analysis provides insight into the substrate recognition and catalytic mechanism of GH31 αGalNAcases.

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.

A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloisomaltooligosaccharides.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(1 → 6)-linked-D-glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest isomaltotetraose or shorter isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an α-(1 → 6)-D-glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.

Sero-diagnostic potential of Plasmodium falciparum recombinant merozoite surface protein (MSP)-3 expressed in silkworm.

Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60-70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.

Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin.

Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.