Characterization of a xylanase belonging to the glycoside hydrolase family 5 subfamily 35 from Paenibacillus sp. H2C.

Corn xylan is resistant to enzymatic hydrolysis due to its complex structure. We characterized PsXyn5A, an enzyme highly active for corn xylan, isolated from Paenibacillus sp. H2C. PsXyn5A is a modular xylanase with a catalytic domain belonging to the glycoside hydrolase family 5 subfamily 35 (GH5_35) and a carbohydrate-binding module family 13 (CBM13) domain. The substrate recognition mechanism of GH5_35 xylanase has not been reported. Analysis of the hydrolysate from rye arabinoxylan (RAX) has shown that the GH5_35 catalytic domain of PsXyn5A recognizes an arabinofuranosyl (Araf) side residue and cleaves the reducing terminal side of Araf-linked xylopyranose. This cleavage specificity is the same as reported for the GH5_34 xylanase from Hungateiclostridium thermocellum (HtXyl5A). Unlike HtXyl5A, PsXyn5A produced Araf-xylopyranose from RAX and did not hydrolyze 33-α-l-Araf-xylotetraose. Deletion of the CBM13 domain significantly decreased the activity toward insoluble corn xylan, indicating that CBM13 plays an essential role in hydrolyzing corn xylan.

Characterization of 3-isopropylmalate dehydrogenase from extremely halophilic archaeon Haloarcula japonica.

3-Isopropylmalate dehydrogenase (IPMDH) catalyzes oxidative decarboxylation of (2R, 3S)-3-isopropylmalate to 2-oxoisocaproate in leucine biosynthesis. In this study, recombinant IPMDH (HjIPMDH) from an extremely halophilic archaeon, Haloarcula japonica TR-1, was characterized. Activity of HjIPMDH increased as KCl concentration increased, and the maximum activity was observed at 3.0 m KCl. Analytical ultracentrifugation revealed that HjIPMDH formed a homotetramer at high KCl concentrations, and it dissociated to a monomer at low KCl concentrations. Additionally, HjIPMDH was thermally stabilized by higher KCl concentrations. This is the first report on haloarchaeal IPMDH.

The mutation of Thr315 to Asn of GH10 xylanase XynR increases the alkaliphily but decreases the alkaline resistance.

XynR is a thermophilic and alkaline GH10 xylanase, identified in the culture broth of alkaliphilic and thermophilic Bacillus sp. strain TAR-1. We previously selected S92E as a thermostable variant from a site saturation mutagenesis library. Here, we attempted to select the alkaliphilic XynR variant from the library and isolated T315N. In the hydrolysis of beechwood xylan, T315N and S92E/T315N exhibited a broader bell-shaped pH-dependent activity than the wild-type (WT) XynR and S92E. The optimal pH values of T315N and S92E/T315N were 6.5-9.5 while those of WT and S92E were 6.5-8.5. On the other hand, T315N and S92E/T315N exhibited a narrower bell-shaped pH dependence of stability: the pHs at which the activity was stable after the incubation at 37 °C for 24 h were 6.0-8.5 for T315N and S92E/T315N, but 6.0-10.0 for WT and S92E. These results indicated that the mutation of Thr315 to Asn increased the alkaliphily but decreased the alkaline resistance.

Functional expression of the reverse transcriptase αß heterodimer from avian myeloblastosis virus in Escherichia coli.

The α subunit of avian myeloblastosis virus reverse transcriptase (AMV-RT) is generated from the ß-subunit by proteolysis, and the αß heterodimer represents the active form. The codon-optimized gene was expressed in Escherichia coli, and an active αß heterodimer was generated. The RNA amplification activity of the purified recombinant AMV-RT αß heterodimer was similar to that of the native one.

Characterization of a GlgC homolog from extremely halophilic archaeon Haloarcula japonica.

Glycogen synthesis in bacteria is mainly organized by the products of glgB, glgC, and glgA genes comprising the widely known glg operon. On the genome of extremely halophilic archaeon Haloarcula japonica, there was a gene cluster analogous to the bacterial glg operon. In this study, we focused on a GlgC homolog of Ha. japonica, and its recombinant enzyme was prepared and characterized. The enzyme showed highest activity toward GTP and glucose-1-phosphate as substrates in the presence of 2.6 m KCl and predicted to be work as "GDP-glucose pyrophosphorylase" in Ha. japonica.

Insight into the mechanism of thermostabilization of GH10 xylanase from Bacillus sp. strain TAR-1 by the mutation of S92 to E.

The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95.

The novel potent TEAD inhibitor, K-975, inhibits YAP1/TAZ-TEAD protein-protein interactions and exerts an anti-tumor effect on malignant pleural mesothelioma.

The Hippo signaling pathway regulates cell fate and organ development. In the Hippo pathway, transcriptional enhanced associate domain (TEAD) which is a transcription factor is activated by forming a complex with yes-associated protein 1 (YAP1) or transcriptional coactivator with PDZ-binding motif (TAZ, also called WWTR1). Hyper-activation of YAP1/TAZ, leading to the activation of TEAD, has been reported in many cancers, including malignant pleural mesothelioma (MPM). Therefore, the YAP1/TAZ-TEAD complex is considered a novel therapeutic target for cancer treatment. However, few reports have described YAP1/TAZ-TEAD inhibitors, and their efficacy and selectivity are poor. In this study, we performed a high-throughput screening of a neurofibromin 2 (NF2)-deficient MPM cell line and a large tumor suppressor kinase 1/2 (LATS1/2)-deficient non-small-cell lung cancer cell line using a transcriptional reporter assay. After screening and optimization, K-975 was successfully identified as a potent inhibitor of YAP1/TAZ-TEAD signaling. X-ray crystallography revealed that K-975 was covalently bound to an internal cysteine residue located in the palmitate-binding pocket of TEAD. K-975 had a strong inhibitory effect against protein-protein interactions between YAP1/TAZ and TEAD in cell-free and cell-based assays. Furthermore, K-975 potently inhibited the proliferation of NF2-non-expressing MPM cell lines compared with NF2-expressing MPM cell lines. K-975 also suppressed tumor growth and provided significant survival benefit in MPM xenograft models. These findings indicate that K-975 is a strong and selective TEAD inhibitor with the potential to become an effective drug candidate for MPM therapy.

Isolation of four xylanases capable of hydrolyzing corn fiber xylan from Paenibacillus sp. H2C.

Corn fibre xylan (CX) shows high resistance to enzymatic hydrolysis due to its densely decorated side chains. To find enzymes capable of hydrolyzing CX, we isolated a bacterial strain (named H2C) from soil, by enrichment culture using non-starch polysaccharides of corn as the sole carbon source. Analysis based on the 16S rRNA sequence placed strain H2C within genus Paenibacillus. Enzymes were purified from supernatant of culture broth of strain H2C based on solubilizing activities toward CX. Four enzymes, Xyn5A, Xyn10B, Xyn11A, and Xyn30A, were successfully identified, which belong to glycoside hydrolase (GH) families, 5, 10, 11, and 30, respectively. Phylogenetic analysis classified Xyn5A in subfamily 35 of GH family 5, a subfamily of unknown function. Their activities toward beechwood xylan and/or wheat arabinoxylan indicated that these enzymes are ß-1,4-xylanases. They showed high solubilizing activities toward a feed material, corn dried distiller's grains with solubles, compared to five previously characterized xylanases.Abbreviations : CX: corn fibre xylan; DDGS: corn dried distiller's grains with solubles.

Increase in the thermostability of GH11 xylanase XynJ from Bacillus sp. strain 41M-1 using site saturation mutagenesis.

GH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a ß-jellyroll fold composed of eight ß strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the ß strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13-Arg104 and Gly169-Tyr194, both of which are located in the ß-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the kcat and Km values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.

Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library.

Site saturation mutagenesis library is a recently developed technique, in which any one out of all amino acid residues in a target region is substituted into other 19 amino acid residues. In this study, we used this technique to increase the thermostability of a GH10 xylanase, XynR, from Bacillus sp. strain TAR-1. We hypothesized that the substrate binding region of XynR is flexible, and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Site saturation mutagenesis libraries of amino acid residues Tyr43-Lys115 and Ala300-Asn325 of XynR were constructed. By screening 480 clones, S92E was selected as the most thermostable one, exhibiting the residual activity of 80% after heat treatment at 80°C for 15 min in the hydrolysis of Remazol Brilliant Blue-xylan. Our results suggest that this strategy is effective for stabilization of GH10 xylanase. ABBREVIATIONS: DNS: 3,5-dinitrosalicylic acid; RBB-xylan: Remazol Brilliant Blue-xylan.

Complete biosynthetic pathway of the C50 carotenoid bacterioruberin from lycopene in the extremely halophilic archaeon Haloarcula japonica.

UNLABELLED: Haloarcula japonica, an extremely halophilic archaeon that requires high concentrations of NaCl for growth, accumulates the C50 carotenoid bacterioruberin (BR). By homology analysis, a gene cluster, including c0507, c0506, and c0505, was found and predicted to be involved in the synthesis of bacterioruberin. To elucidate the function of the encoded enzymes, we constructed Ha. japonica mutants of these genes and analyzed carotenoids produced by the mutants. Our research showed that c0507, c0506, and c0505 encoded a carotenoid 3,4-desaturase (CrtD), a bifunctional lycopene elongase and 1,2-hydratase (LyeJ), and a C50 carotenoid 2",3"-hydratase (CruF), respectively. The above three carotenoid biosynthetic enzymes catalyze the reactions that convert lycopene to bacterioruberin in Ha. japonica. This is the first identification of functional CrtD and CruF in archaea and elucidation of the complete biosynthetic pathway of bacterioruberin from lycopene. IMPORTANCE: Haloarcula japonica, an extremely halophilic archaeon, accumulates the C50 carotenoid bacterioruberin (BR). In this study, we have identified three BR biosynthetic enzymes and have elucidated their functions. Among them, two enzymes were found in an archaeon for the first time. Our results revealed the biosynthetic pathway responsible for production of BR in Ha. japonica and provide a basis for investigating carotenoid biosynthetic pathways in other extremely halophilic archaea. Elucidation of the carotenoid biosynthetic pathway in Ha. japonica may also prove useful for producing the C50 carotenoid BR efficiently by employing genetically modified haloarchaeal strains.

Identification of carotenoids from the extremely halophilic archaeon Haloarcula japonica.

The carotenoids produced by extremely halophilic archaeon Haloarcula japonica were extracted and identified by their chemical, chromatographic, and spectroscopic characteristics (UV-Vis and mass spectrometry). The composition (mol%) was 68.1% bacterioruberin, 22.5% monoanhydrobacterioruberin, 9.3% bisanhydrobacterioruberin, <0.1% isopentenyldehydrorhodopin, and trace amounts of lycopene and phytoene. The in vitro scavenging capacity of a carotenoid, bacterioruberin, extracted from Haloarcula japonica cells against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was evaluated. The antioxidant capacity of bacterioruberin was much higher than that of ß -carotene.

Gene analysis, expression, and characterization of an intracellular α-amylase from the extremely halophilic archaeon Haloarcula japonica.

Haloarcula japonica is an extremely halophilic archaeon that requires high concentrations of NaCl to grow. Recently the draft genome sequence of Ha. japonica was determined, and a gene encoding an α-amylase, malA, was identified. The deduced amino acid sequence of MalA, consisting of 663 amino acids, showed homology to α-amylase family enzymes. The sequence did not contain a secretion signal sequence, indicating that the protein is a cytoplasmic enzyme. Transcription of the malA gene was confirmed by reverse transcription (RT)-PCR, and the transcription start site was determined by a 5'-RACE experiment. The malA gene was cloned and expressed in Ha. japonica. The recombinant MalA was purified and characterized. MalA required a high concentration of NaCl for starch-hydrolyzing activity. It showed higher activity on soluble starch, amylose, and amylopectin, and lower activity on glycogen.

Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli.

Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP-HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP-HepI offered a 30.6% increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca2+ as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP-HepI showed a specific activity of 56.3 IU/mg, 206% higher than that of the wild type obtained without Ca2+ addition, along with a 177% increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP-HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.

Mutational analysis of a CBM family 5 chitin-binding domain of an alkaline chitinase from Bacillus sp. J813.

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.

Biochemical analysis and kinetic modeling of the thermal inactivation of MBP-fused heparinase I: implications for a comprehensive thermostabilization strategy.

Enzymatic degradation of heparin by heparin lyases has not only largely facilitated heparin structural analysis and contamination detection, but also showed great potential to be a green and cost-effective way to produce low molecular weight heparin (LMWH). However, the commercial use of heparinase I (HepI), one of the most studied heparin lyases, has been largely hampered by its low productivity and extremely poor thermostability. Here we report the thermal inactivation mechanism and strategic thermal stabilization of maltose-binding protein (MBP)-HepI, a fusion HepI produced in E. coli with high yield, solubility and activity. Biochemical studies demonstrated that the thermal inactivation of MBP-HepI involves an unfolding step that is temperature-dependently reversible, followed by an irreversible dimerization step induced by intermolecular disulfide bonds. A good consistency between the kinetic modeling and experimental data of the inactivation was obtained within a wide range of temperature and enzyme concentration, confirming the adequacy of the proposed inactivation model. Based on the inactivation mechanism, a comprehensive strategy was proposed for the thermal stabilization of MBP-HepI, in which Ca(2+) and Tween 80 were used to inhibit unfolding while site mutation at Cys297 and DTT were employed to suppress dimerization. The engineered enzyme exhibits remarkably improved storage and operational thermostability, for example, 16-fold increase in half-life at its optimum temperature of 30 °C and 8-fold increase in remaining activity of 95% after 1-week storage at 4 °C, and therefore shows great potential as a commercial biocatalyst for heparin degradation in the pharmaceutical industry.

A calcium-dependent xylan-binding domain of alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1.

Xylanase J of alkaliphilic Bacillus sp. strain 41M-1 contains a carbohydrate-binding module family 36 xylan-binding domain (XBD). Mutational analysis of the XBD revealed that Tyr237, Asp313, Trp317, and Asp318 were involved in Ca(2+)-dependent xylan-binding, and that Asp313 and Asp318 were especially important.

Role of exposed aromatic residues in substrate-binding of CBM family 5 chitin-binding domain of alkaline chitinase.

Chitinase J (ChiJ) from alkaliphilic Bacillus sp. strain J813 has a multidomain structure containing a catalytic domain (CatD), a fibronectin type III like domain (FnIIID) and a chitin-binding domain (ChBD). It has been shown that the ChBD binds to an insoluble chitin and enhances its degradation by the CatD. Further binding study of the ChBD was performed with a glutathione-S-transferase fusion protein. This fusion protein showed binding abilities to insoluble chitin and chitosan. Two surface-exposed aromatic residues (Trp541 and Trp542) were found in the tertiary-structure model of ChBD and targeted for mutational analysis. Single and double mutations of the two aromatic residues decreased the chitin- and chitosan-binding abilities. It was revealed that these residues would be important for substrate-binding of the ChBD.

Additional carbohydrate-binding modules enhance the insoluble substrate-hydrolytic activity of beta-1,3-glucanase from alkaliphilic Nocardiopsis sp. F96.

Beta-1,3-glucanase (BglF) from Nocardiopsis sp. F96 is composed of only a catalytic domain. To improve the enzymatic properties of BglF, we attempted to construct chimeric enzymes consisting of BglF and some carbohydrate-binding modules, such as the C-terminal additional domain (CAD) and the N-terminal additional domain (NAD) of beta-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain (ChBD) of chitinase from alkaliphilic Bacillus sp. J813. CAD-fused BglF (BglF-CAD), NAD-fused BglF (NAD-BglF), both NAD- and CAD-fused BglF (NAD-BglF-CAD) and ChBD-fused BglF (BglF-ChBD) were constructed and characterized. The addition of CAD caused increases in binding abilities and hydrolytic activities toward insoluble beta-1,3-glucans. As well as BglF-CAD, the binding ability and hydrolytic activity of BglF-ChBD toward pachyman were also increased. The hydrolytic activity of BglF-CAD at pH 9-10 was higher than that of BglF. The relative activities of BglF-CAD and BglF-ChBD at around 50-70 degrees C were higher than that of BglF.

Improvement of alkaliphily of Bacillus alkaline xylanase by introducing amino acid substitutions both on catalytic cleft and protein surface.

Xylanase J (XynJ) from alkaliphilic Bacillus sp. 41M-1 is an alkaline xylanase. The crystal structure has been solved with XynJ. Improvement of the alkaliphily of XynJ was attempted by amino acid substitutions. Reinforcing the characteristic salt bridge in the catalytic cleft and introducing excess Arg residues on the protein surface shifted the optimum pH of the wild-type enzyme from 8.5 to 9.5.