Isolation of Pro-Osteogenic Compounds from Euptelea polyandra That Reciprocally Regulate Osteoblast and Osteoclast Differentiation.

Plants contain a large number of small-molecule compounds that are useful for targeting human health and in drug discovery. Healthy bone metabolism depends on the balance between bone-forming osteoblast activity and bone-resorbing osteoclast activity. In an ongoing study searching for 22 plant extracts effective against osteoporosis, we found that the crude extract of Euptelea polyandra Sieb. et Zucc (E. polyandra) had osteogenic bioactivity. In this study, we isolated two compounds, isoquercitrin (1) and astragalin (2), responsible for osteogenic bioactivity in osteoblastic MC3T3-E1 cells from the leaf of E. polyandra using column chromatography and the spectroscopic technique. This is the first report to isolate astragalin from E. polyandra. Compounds (1) and (2) promoted osteoblast differentiation by increasing alkaline phosphatase (ALP) activity and alizarin red S stain-positive calcium deposition, while simultaneously suppressing tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation in RAW264.7 cells at non-cytotoxic concentrations. Isoquercitrin (1) and astragalin (2) increased the expression of osteoblastic differentiation genes, Osterix, ALP, and Osteoprotegerin in the MC3T3-E1 cells, while suppressing osteoclast differentiation genes, TRAP, Cathepsin K, and MMP 9 in the RAW264.7 cells. These compounds may be ideal targets for the treatment of osteoporosis due to their dual function of promoting bone formation and inhibiting bone resorption.

1H-NMR-Based Metabolomics for the Classification of the Roots of Paeonia lactiflora, a Constituent of Kampo Medicines.

The root of Paeonia lactiflora (PAEONIAE RADIX) is a constituent of the traditional Japanese medicines (Kampo) and is known to have various effects. Peony roots cultivated in Japan and China are available in the Japanese market for medicinal use. In this study, the chemical diversity of ten available peony roots in the market that differed in their cultivation area was investigated using 1H-NMR metabolomics techniques. Principal component analysis and hierarchical cluster analysis of the 1H-NMR spectra of the peony roots methanolic extracts revealed a clear difference between the metabolic profiles of Japanese and Chinese peony roots. By preparative procedures using chromatography based on 1H-NMR spectra measurements, oxypaeoniflorin and (+)-catechin were found to be specific compounds for Japanese peony root. All peony roots used in this study were listed in the Japanese Pharmacopoeia. Therefore, the differences in the constituents of these peony roots might be attributed to growing conditions than differences in species. Cultivation conditions also influence the quality of natural medicines.

Degradative Effect of Nattokinase on Spike Protein of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic and has inflicted enormous damage on the lives of the people and economy of many countries worldwide. However, therapeutic agents against SARS-CoV-2 remain unclear. SARS-CoV-2 has a spike protein (S protein), and cleavage of the S protein is essential for viral entry. Nattokinase is produced by Bacillus subtilis var. natto and is beneficial to human health. In this study, we examined the effect of nattokinase on the S protein of SARS-CoV-2. When cell lysates transfected with S protein were incubated with nattokinase, the S protein was degraded in a dose- and time-dependent manner. Immunofluorescence analysis showed that S protein on the cell surface was degraded when nattokinase was added to the culture medium. Thus, our findings suggest that nattokinase exhibits potential for the inhibition of SARS-CoV-2 infection via S protein degradation.

Cyanobacterial branching enzymes bind to α-glucan via surface binding sites.

Besides their catalysis, specific interactions between starch/glycogen processing enzymes and their substrates have been reported. Multiple branching enzyme (BE) isoforms, BE1, BE2, and BE3, have been found in a limited number of cyanobacterial species that are characterized by amylopectin accumulation. Seven surface binding sites (SBSs) located away from the active site have been identified in crystal structures of cyanobacterial BE1 from Crocosphaera subtropica (Cyanothece sp.) ATCC 51142 (51142BE1). In the present study, binding affinity toward amylopectin, amylose, and glycogen was investigated for wild-type 51142BE1 and its mutants (residues at SBSs important for sugar-binding were replaced by alanine). These enzymes showed retarded mobility during electrophoresis in non-denaturing polyacrylamide gels in the presence of polysaccharides. This was caused by interactions between the enzymes and the polysaccharides, enabling calculation of the dissociation constants (Kd values) of the enzymes toward the polysaccharides. Mutational analysis indicated that particular domains of the protein (domains A and C) were involved in the polysaccharide binding. Kd values toward the polysaccharides were also measured for 10 BE isoforms (five BE1, three BE2, and two BE3) from 5 cyanobacterial strains. All BEs displayed much lower Kd values (higher affinity) toward amylopectin and amylose than toward glycogen, as described for plant BEs. In addition, one BE2 displayed exceptionally high Kd values (low affinity), while two BE3 exhibited multiple Kd values to all polysaccharides. These results could be ascribed to sequence variations in the SBSs, irrespective of the catalytic specificity.

Use of 13C-NMR Chemical Shifts; Application of Principal Component Analysis for Categorizing Structurally Similar Methoxyflavones and Correlation Analysis between Chemical Shifts and Cytotoxicity.

The 13C-NMR spectral data for the 15-carbon flavonoid skeleton in eleven methoxyflavones isolated from Kaempferia parviflora (Zingiberaceae) were processed by principal component analysis (PCA). Based on the PCA score plots, the methoxyflavones were categorized into three groups according to their structural features. The cytotoxicities of the methoxyflavones toward 3T3-L1 murine preadipocyte cells were evaluated by 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and found to differ according to structure. The relationship between the 13C-NMR chemical shifts of the methoxyflavones and their cytotoxicities was investigated using Pearson's correlation analysis. The 13C-NMR signal at C-10, a quaternary carbon, was correlated with cytotoxicity. Based on these results, a structural design which lowers the 13C-NMR chemical shift at C-10 would be important for the development of cytotoxic compounds. Although quantitative structure-activity and structure-property relationships are well established paradigms for predicting trends among a series of compounds, quantitative property-activity relationships have been relatively unstudied. This approach offers a new strategy for directing structure-activity relationship research.

Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model.

Branching enzyme (BE) catalyzes the formation of α-1,6-glucosidic linkages in amylopectin and glycogen. The reaction products are variable, depending on the organism sources, and the mechanistic basis for these different outcomes is unclear. Although most cyanobacteria have only one BE isoform belonging to glycoside hydrolase family 13, Cyanothece sp. ATCC 51142 has three isoforms (BE1, BE2, and BE3) with distinct enzymatic properties, suggesting that investigations of these enzymes might provide unique insights into this system. Here, we report the crystal structure of ligand-free wild-type BE1 (residues 5-759 of 1-773) at 1.85 Å resolution. The enzyme consists of four domains, including domain N, carbohydrate-binding module family 48 (CBM48), domain A containing the catalytic site, and domain C. The central domain A displays a (ß/α)8-barrel fold, whereas the other domains adopt ß-sandwich folds. Domain N was found in a new location at the back of the protein, forming hydrogen bonds and hydrophobic interactions with CBM48 and domain A. Site-directed mutational analysis identified a mutant (W610N) that bound maltoheptaose with sufficient affinity to enable structure determination at 2.30 Å resolution. In this structure, maltoheptaose was bound in the active site cleft, allowing us to assign subsites -7 to -1. Moreover, seven oligosaccharide-binding sites were identified on the protein surface, and we postulated that two of these in domain A served as the entrance and exit of the donor/acceptor glucan chains, respectively. Based on these structures, we propose a substrate binding model explaining the mechanism of glycosylation/deglycosylation reactions catalyzed by BE.

Cesium Uptake by Rice Roots Largely Depends Upon a Single Gene, HAK1, Which Encodes a Potassium Transporter.

Incidents at the Fukushima and Chernobyl nuclear power stations have resulted in widespread environmental contamination by radioactive nuclides. Among them, 137cesium has a 30 year half-life, and its persistence in soil raises serious food security issues. It is therefore important to prevent plants, especially crop plants, from absorbing radiocesium. In Arabidopsis thaliana, cesium ions are transported into root cells by several different potassium transporters such as high-affinity K+ transporter 5 (AtHAK5). Therefore, the cesium uptake pathway is thought to be highly redundant, making it difficult to develop plants with low cesium uptake. Here, we isolated rice mutants with low cesium uptake and reveal that the Oryza sativa potassium transporter OsHAK1, which is expressed on the surfaces of roots, is the main route of cesium influx into rice plants, especially in low potassium conditions. During hydroponic cultivation with low to normal potassium concentrations (0-206 µM: the normal potassium level in soil), cesium influx in OsHAK1-knockout lines was no greater than one-eighth that in the wild type. In field experiments, knockout lines of O. sativa HAK1 (OsHAK1) showed dramatically reduced cesium concentrations in grains and shoots, but their potassium uptake was not greatly affected and their grain yields were similar to that of the wild type. Our results demonstrate that, in rice roots, potassium transport systems other than OsHAK1 make little or no contribution to cesium uptake. These results show that low cesium uptake rice lines can be developed for cultivation in radiocesium-contaminated areas.

Establishment of a Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method and Its Application to Lichen Profiling.

Direct-injection electron ionization-mass spectrometry (DI-EI-MS) is a multivariate analysis method useful for characterizing biological materials. We demonstrated the use of DI-EI-MS for metabolic profiling using several closely related lichen species: Cladonia krempelhuberi, C. gracilis, C. pseudogymnopoda, and C. ramulosa. The methodology involves conversion of total ion chromatograms to integrated chromatograms and assessment of reproducibility. The qualitative DI-EI-MS method was used to profile the major and/or minor constituents in extracts of lichen samples. It was possible to distinguish each lichen sample by altering the electron energy in DI-EI-MS and examining the resulting data using one-way analysis of variance. Previously undetectable peaks, which are easy to fragment could be revealed by varying the electron energy. Our results suggest that metabolic profiling using DI-EI-MS would be useful for discriminating between subgroups within the same species. This is the first study to report the use of DI-EI-MS in a metabolomics application.

Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1 → 4)-glucan.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1 â†’ 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1 â†’ 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1 â†’ 4)-> α-(1 â†’ 2)- > α-(1 â†’ 3)- > α-(1 â†’ 6)-glucobiose and maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by 13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1 â†’ 4) to α-(1 â†’ 6) transglucosylation activity. Ps6GT31A elongated α-(1 â†’ 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1 â†’ 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

Distribution of glucan-branching enzymes among prokaryotes.

Glucan-branching enzyme plays an essential role in the formation of branched polysaccharides, glycogen, and amylopectin. Only one type of branching enzyme, belonging to glycoside hydrolase family 13 (GH13), is found in eukaryotes, while two types of branching enzymes (GH13 and GH57) occur in prokaryotes (Bacteria and Archaea). Both of these types are the members of protein families containing the diverse specificities of amylolytic glycoside hydrolases. Although similarities are found in the catalytic mechanism between the two types of branching enzyme, they are highly distinct from each other in terms of amino acid sequence and tertiary structure. Branching enzymes are found in 29 out of 30 bacterial phyla and 1 out of 5 archaeal phyla, often along with glycogen synthase, suggesting the existence of α-glucan production and storage in a wide range of prokaryotes. Enormous variability is observed as to which type and how many copies of branching enzyme are present depending on the phylum and, in some cases, even among species of the same genus. Such a variation may have occurred through lateral transfer, duplication, and/or differential loss of genes coding for branching enzyme during the evolution of prokaryotes.

p-Coumaroyl Malate Derivatives of the Pandanus amaryllifolius Leaf and Their Isomerization.

A novel p-coumaroyl dimethyl malate (1) was isolated from the Pandanus amaryllifolius leaf in addition to three known analogs of p-coumaroyl dimethyl malate (2-4), and their structures were elucidated by analysis of the spectroscopic data. The p-coumaroyl malate derivatives were isolated as a mixture of E and Z isomers. To determine the cause of isomerization, the p-coumaroyl malate isolated in this study was synthesized. We concluded that the Z isomer might be an artifact generated from the E isomer through purification steps.

Functional characterization of three (GH13) branching enzymes involved in cyanobacterial starch biosynthesis from Cyanobacterium sp. NBRC 102756.

Starch and glycogen are widespread storage polysaccharides in bacteria, plants, and animals. Recently, some cyanobacteria were found to accumulate water-insoluble α-glucan similar to amylopectin rather than glycogen, the latter of which is more commonly produced in these organisms. The amylopectin-producing species including Cyanobacterium sp. NBRC 102756 invariably have three branching enzyme (BE) homologs, BE1, BE2, and BE3, all belonging to the glycoside hydrolase family 13. Multiple BE isoforms in prokaryotes have not been previously studied. In the present work, we carried out functional characterization of these enzymes expressed in Escherichia coli. The recombinant enzymes were all active, although the specific activity of BE3 was much lower than those of BE1 and BE2. After the incubation of the enzymes with amylopectin or amylose, the reaction products were analyzed by fluorophore-assisted carbohydrate capillary electrophoresis method. BE1 and BE2 showed similar chain-length preference to BEIIb isoform of rice (Oryza sativa L.), while the catalytic specificity of BE3 was similar to that of rice BEI. These results indicate that starch-producing cyanobacteria have both type-I BE (BE3) and type-II BEs (BE1 and BE2) in terms of chain-length preferences, as is the case of plants. All BE isoforms were active against phosphorylase limit dextrin, in which outer branches had been uniformly diminished to 4 glucose residues. Based on its catalytic properties, BE3 was assumed to have a role to transfer the glucan chain bearing branch(es) to give rise to a newly growing unit, or cluster as observed in amylopectin molecule.

Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity.

Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of glycoside hydrolase family 66 and it produces cycloisomaltooligosaccharides (CIs). Small CIs (CI-7-9) and large CIs (CI-≥10) are designated as oligosaccharide-type CIs (oligo-CIs) and megalosaccharide-type CIs (megalo-CIs) respectively. CITase from Bacillus circulans T-3040 (BcCITase) produces mainly CI-8 with little megalo-CIs. It has two family 35 carbohydrate-binding modules (BcCBM35-1 and BcCBM35-2). BcCBM35-1 is inserted in a catalytic domain of BcCITase and BcCBM35-2 is located at the C-terminal region. Our previous studies suggested that BcCBM35-1 has two substrate-binding sites (B-1 and B-2) [Suzuki et al. (2014) J. Biol. Chem. 289, 12040-12051]. We implemented site-directed mutagenesis of BcCITase to explore the preference for product size on the basis of the 3D structure of BcCITase. Mutational studies provided evidence that B-1 and B-2 contribute to recruiting substrate and maintaining product size respectively. A mutant (mutant-R) with four mutations (F268V, D469Y, A513V and Y515S) produced three times as much megalo-CIs (CI-10-12) and 1.5 times as much total CIs (CI-7-12) as compared with the wild-type (WT) BcCITase. The 3D structure of the substrate-enzyme complex of mutant-R suggested that the modified product size specificity was attributable to the construction of novel substrate-binding sites in the B-2 site of BcCBM35-1 and reactivity was improved by mutation on subsite -3 on the catalytic domain.

Structural elucidation of the cyclization mechanism of α-1,6-glucan by Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase.

Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (ß/α)8-barrel domain and three ß-domains. Domain N with an immunoglobulin-like ß-sandwich fold was attached to the N terminus; domain C with a Greek key ß-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) ß-jellyroll domain B was inserted between the 7th ß-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite -8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.

Crystal structures of a glycoside hydrolase family 20 lacto-N-biosidase from Bifidobacterium bifidum.

Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-ß1,3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 ß-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-ß1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique ß-trefoil-like fold. Compared with other ß-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a -2 subsite specific for ß-1,3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near (4)E, whereas that of LNB-thiazoline is in a (4)C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed.

Evidence for cycloisomaltooligosaccharide production from starch by Bacillus circulans T-3040.

Bacillus circulans T-3040 produces cycloisomaltooligosaccharide glucanotransferase (CITase) and cycloisomaltooligosaccharides (cyclodextrans, CIs) when it is grown in media containing dextran as the carbon source. To investigate the effects of carbon sources on CITase activity, B. circulans T-3040 was cultured with glucose; sucrose; a mixture of isomaltose, isomaltotriose, and panose (IMOs); a mixture of maltohexaose and maltoheptaose (G67); dextrin (average degree of polymerization = 36); dextran 40; and soluble starch. In addition to dextran 40, CIs were produced when the T-3040 strain was grown in media containing soluble starch as the sole carbon source. CITase production was induced by dextran 40, IMOs, and soluble starch but not by G67 or dextrin, which suggests that α-1,6 glucosidic linkages are required for CITase induction. Although CITase was induced by IMOs, no CIs were produced in the culture. CI-producing activity in the presence of soluble starch as the substrate (SS-CITase activity) was observed only in cultures containing dextran 40 or soluble starch. The production of CITase was significantly unaffected by glucose addition, but SS-CITase activity almost completely disappeared after glucose addition. A 135-kDa protein was found to contribute to CI formation from starch in the presence of CITase. This protein had a disproportionation activity with maltooligosaccharides, and its induction and inhibition system may be different from those of CITase.

Inhibitory effects of senkyuchachosan on SARS-CoV-2 papain-like protease activity in vitro.

Papain-like protease (PLpro) enzyme plays a vital role in viral replication as it breaks down polyproteins and disrupts the host's immune response. There are few reports on Kampo formulas that focus on PLpro activity. In this study, we evaluated the inhibitory effects of senkyuchachosan, a traditional Japanese medicine, on PLpro of SARS-CoV-2, the virus responsible for causing COVID-19. We purified the PLpro enzyme and conducted in vitro enzymatic assays using specific substrates. Among the nine crude drugs present in senkyuchachosan, four (Cyperi Rhizoma, Schizonepetae Spica, Menthae Herba, and Camelliae sinensis Folium [CsF]) strongly inhibited PLpro activity. CsF, derived from Camellia sinensis (green tea), contains polyphenols, including catechins and tannins. To confirm that the PLpro inhibitory effects of senkyuchachosan predominantly stem from tannins, the tannins were removed from the decoction using polyvinylpolypyrrolidone (PVPP). The inhibitory effect of senkyuchachosan on PLpro activity was reduced by the removal of PVPP. In addition, the tannin fraction obtained from the CsF extracts showed significant PLpro inhibitory effects. These findings lay the groundwork for the potential development of therapeutic agents that target SARS-CoV-2 infection by intervening in proteolytic cleavage of the virus.

Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach.

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7-14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr(451)-Val(1082)), a portion of which shares identity (35% at Ala(39)-Ser(1304) of PsDex) with Pro(32)-Ala(755) of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val(837)-Met(932) for PsDex and Tyr(404)-Tyr(492) for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala(39)-Ser(1304)) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp(189), Asp(340), Glu(412), and Asp(1254) of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1,500 to 1/40,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN(3). D340G or E412Q formed a ß- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp(340) and Glu(412) as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN(3).

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K.

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the k(cat)/K(M) value of CITase-598K was approximately two times higher (32.2s(-1)mM(-1)) than that of CITase-T3040 (17.8s(-1)mM(-1)). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.

Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation.

Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.