C-terminal Carbohydrate-binding Module Research Articles

Structural and binding studies of a new chitin-active AA10 lytic polysaccharide monooxygenase from the marine bacterium Vibrio campbellii.

Vibrio spp. play a crucial role in the global recycling of the highly abundant recalcitrant biopolymer chitin in marine ecosystems through their ability to secrete chitin-degrading enzymes to efficiently hydrolyse chitinous materials and use them as their major carbon source. In this study, the first crystal structures of a complete four-domain chitin-active AA10 lytic polysaccharide monooxygenase from the chitinolytic bacterium Vibrio campbellii type strain ATCC BAA-1116 are reported. The crystal structures of apo and copper-bound VhLPMO10A were resolved as homodimers with four distinct domains: an N-terminal AA10 catalytic (CatD) domain connected to a GlcNAc-binding (GbpA_2) domain, followed by a module X domain and a C-terminal carbohydrate-binding module (CBM73). Size-exclusion chromatography and small-angle X-ray scattering analysis confirmed that VhLPMO10A exists as a monomer in solution. The active site of VhLPMO10A is located on the surface of the CatD domain, with three conserved residues (His1, His98 and Phe170) forming the copper(II)-binding site. Metal-binding studies using synchrotron X-ray absorption spectroscopy and X-ray fluorescence, together with electron paramagnetic resonance spectroscopy, gave consistently strong copper(II) signals in the protein samples, confirming that VhLPMO10A is a copper-dependent enzyme. ITC binding data showed that VhLPMO10A could bind various divalent cations but bound most strongly to copper(II) ions, with a Kd of 0.1 ± 0.01 µM. In contrast, a Kd of 1.9 nM was estimated for copper(I) ions from redox-potential measurements. The presence of ascorbic acid is essential for H2O2 production in the reaction catalysed by VhLPMO10A. MALDI-TOF MS identified VhLPMO10A as a C1-specific LPMO, generating oxidized chitooligosaccharide products with different degrees of polymerization (DP2ox-DP8ox). This new member of the chitin-active AA10 LPMOs could serve as a powerful biocatalyst in biofuel production from chitin biomass.

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks.

Microbial expansin-related proteins are ubiquitous across bacterial and fungal organisms and reportedly play a role in the modification and deconstruction of cell wall polysaccharides, including lignocellulose. So far, very few microbial expansin-related proteins, including loosenins and loosenin-like (LOOL) proteins, have been functionally characterized. Herein, four LOOLs encoded by Phanerochaete carnosa and belonging to different subfamilies (i.e., PcaLOOL7 and PcaLOOL9 from subfamily A and PcaLOOL2 and PcaLOOL12 from subfamily B) were recombinantly produced and the purified proteins were characterized using diverse cellulose and chitin substrates. The purified PcaLOOLs weakened cellulose filter paper and cellulose nanofibril networks (CNF); however, none significantly boosted cellulase activity on the selected cellulose substrates (Avicel and Whatman paper). Although fusing the family 63 carbohydrate-binding module (CBM63) of BsEXLX1 encoded by Bacillus subtilis to PcaLOOLs increased their binding to cellulose, the CBM63 fusion appeared to reduce the cellulose filter paper weakening observed using wild-type proteins. Binding of PcaLOOLs to alpha-chitin was considerably higher than that to cellulose (Avicel) and was pH dependent, with the highest binding at pH 5.0. Amendment of certain PcaLOOLs in fungal liquid cultivations also impacted the density of the cultivated mycelia. The present study reveals the potential of fungal expansin-related proteins to impact both cellulose and chitin networks and points to a possible biological role in fungal cell wall processing. IMPORTANCE The present study deepens investigations of microbial expansin-related proteins and their applied significance by (i) reporting a detailed comparison of diverse loosenins encoded by the same organism, (ii) considering both cellulosic and chitin-containing materials as targeted substrates, and (iii) investigating the impact of the C-terminal carbohydrate binding module (CBM) present in other expansin-related proteins on loosenin function. By revealing the potential of fungal loosenins to impact both cellulose and chitin-containing networks, our study reveals a possible biological and applied role of loosenins in fungal cell wall processing.

Carbohydrate-Binding Module and Linker Allow Cold Adaptation and Salt Tolerance of Maltopentaose-Forming Amylase From Marine Bacterium Saccharophagus degradans 2-40 T.

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40T. We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

The glucoamylase from Aspergillus wentii: Purification and characterization.

This study describes for the first time the purification and characterization of a glucoamylase from Aspergillus wentii (strain PG18), a species of the Aspergillus genus Cremei section. Maximum enzyme production (∼3.5 U/ml) was obtained in submerged culture (72 h) with starch as the carbon source, at 25°C, and with orbital agitation (100 rpm). The enzyme was purified with one-step molecular exclusion chromatography. The 86 kDa purified enzyme hydrolyzed starch in a zymogram and had activity against p-nitrophenyl α- d-glucopyranoside. The optimal enzyme pH and temperature were 5.0 and 60°C (at pH 5.0), respectively. The Tm of the purified enzyme was 60°C, at pH 7.0. The purified glucoamylase had a KM for starch of 1.4 mg/ml and a Vmax of 0.057 mg/min of hydrolyzed starch. Molybdenum activated the purified enzyme, and sodium dodecyl sulfate inhibited it. A thin layer chromatography analysis revealed glucose as the enzyme's main starch hydrolysis product. An enzyme's peptide sequence was obtained by mass spectrometry and used to retrieve a glucoamylase within the annotated genome of A. wentii v1.0. An in silico structural model revealed a N-terminal glycosyl hydrolases family 15 (GH15) domain, which is ligated by a linker to a C-terminal carbohydrate-binding module (CBM) from the CBM20 family.

Specific hydrolysis of curdlan with a novel glycoside hydrolase family 128 β-1,3-endoglucanase containing a carbohydrate-binding module

The cbm6e gene from Saccharophagus degradans 2-40 T was cloned and expressed in Escherichia coli. CBM6E contains a glycoside hydrolase family 128 (GH128) catalytic module and a C-terminal carbohydrate-binding module (CBM) grouped into CBM family 6. The purified recombinant CBM6E displayed high substrate specificity toward curdlan as an endo-β-1,3-glucanase and had maximal activity at pH 6.0 and 35 ℃. The hydrolytic products against curdlan were predominantly laminaritriose (L3) and laminaritetraose (L4) along with a lower amount of laminaripentaose (L5) and laminarihexaose (L6). The CBM6 module selectively enhanced the enzyme activity against curdlan and displayed strict binding specificity to curdlan, no matter in its powder or high-set gel forms. This study laid a foundation for enzymatic degradation of curdlan to produce high-value β-1,3-glucooligosaccharides at moderate temperatures and provided a novel CBM tag for enzyme immobilization on curdlan.

Novel Endotype Xanthanase from Xanthan-Degrading Microbacterium sp. Strain XT11.

Under general aqueous conditions, xanthan appears in an ordered conformation, which makes its backbone largely resistant to degradation by known cellulases. Therefore, the xanthan degradation mechanism is still unclear because of the lack of an efficient hydrolase. Here, we report the catalytic properties of MiXen, a xanthan-degrading enzyme identified from the genus Microbacterium MiXen is a 952-amino-acid protein that is unique to strain XT11. Both the sequence and structural features suggested that MiXen belongs to a new branch of the GH9 family and has a multimodular structure in which a catalytic (α/α)6 barrel is flanked by an N-terminal Ig-like domain and by a C-terminal domain that has very few homologues in sequence databases and functions as a carbohydrate-binding module (CBM). Based on circular dichroism, shear-dependent viscosity, and reducing sugar and gel permeation chromatography analysis, we demonstrated that recombinant MiXen efficiently and randomly cleaved glucosidic bonds within the highly ordered xanthan substrate. A MiXen mutant free of the C-terminal CBM domain partially lost its xanthan-hydrolyzing ability because of decreased affinity toward xanthan, indicating the CBM domain assisted MiXen in hydrolyzing highly ordered xanthan via recognizing and binding to the substrate. Furthermore, side chain substituents and the terminal mannosyl residue significantly influenced the activity of MiXen via the formation of barriers to enzymolysis. Overall, the results of this study provide insight into the hydrolysis mechanism and enzymatic properties of a novel endotype xanthanase that will benefit future applications.IMPORTANCE This work characterized a novel endotype xanthanase, MiXen, and elucidated that the C-terminal carbohydrate-binding module of MiXen could drastically enhance the hydrolysis activity of the enzyme toward highly ordered xanthan. Both the sequence and structural analysis demonstrated that the catalytic domain and carbohydrate-binding module of MiXen belong to the novel branch of the GH9 family and CBMs, respectively. This xanthan cleaver can help further reveal the enzymolysis mechanism of xanthan and provide an efficient tool for the production of molecular modified xanthan with new physicochemical and physiological functions.

Improvement of the catalytic characteristics of a salt-tolerant GH10 xylanase from Streptomyce rochei L10904

A GH10 xylanase Srxyn10 from Streptomyce rochei L10904, and its truncated derivative, Srxyn10M, were investigated. Both displayed great salt-tolerant ability, retaining more than 95% and 91% activity after incubation at 37°C for 1h in 3.0M and 5.0M NaCl, respectively. They exhibited a special hydrolytic property of forming xylobiose as the major product and produced fewer xylose compounds when combined with a reported xylanase while digesting corncob xylans. The mutant, Srxyn10M, was constructed from Srxyn10 by deleting the C-terminal carbohydrate-binding module. It possessed a 3.26-fold higher specific activity on beechwood xylan than Srxyn10. Moreover, Srxyn10M showed greater substrate affinity and catalytic efficiency than Srxyn10 when beechwood xylan, birchwood xylan, and oat-spelt xylan were used as substrates. The thermostability was also greatly improved. Therefore, the application potential was markedly enhanced by the improvement of these properties.

Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules.

Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1→4)-2-acetamido-2-deoxy-β-D-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N'-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-D-glucosaminide (1→4)-β-linkages and are thus "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2°C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23–25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don’t show homology to each other, but all of them are built up from 3–6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70–75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9–1.7mM of KM and 80–120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.

A novel member of family 30 glycoside hydrolase subfamily 8 glucuronoxylan endo-β-1,4-xylanase (CtXynGH30) from Clostridium thermocellum orchestrates catalysis on arabinose decorated xylans

A thermophilic enzyme, CtXynGH30 from Clostridium thermocellum was identified as glucuronoxylan endo-β-1,4-xylanase displayed activity extending to arabinose decorated xylans unlike other glucuronoxylan endo-β-1,4-xylanase from family 30 sub-family 8 of glycoside hydrolase. Modular CtXynGH30 comprises N-terminal catalytic module CtXyn30A and C-terminal carbohydrate binding module of family 6, (CtCBM6). The purified CtXynGH30 displayed a molecular mass of approximately, 60kDa. CtXynGH30 showed an optimum pH 6.0 and optimum temperature 70°C. CtXynGH30 displayed maximum activity against glucuronic acid substituted beechwood xylan followed by birchwood- and 4-O-methyl glucurono-xylan. CtXynGH30 also displayed activity against arabinoxylans that were also confirmed by TLC analysis. The ability of CtXynGH30 to hydrolyse both glucuronic and arabinose decorated xylans distinguishes it from other enzymes of this subfamily. The 1H NMR analysis of hydrolysed products of beechwood xylan confirmed the presence of acidic xylo-oligosaccharides having methyl glucuronic acid moiety penultimate to the reducing end of xylan. CtXynGH30 produced a range of acidic xylo-oligosaccharides and arabinoxylo-oligosaccharides which have potential food and health applications.

An Enzymatically Active β-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family.

Endo-β-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic β-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that β-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen β-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long β-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of β-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.

The effect of the carbohydrate binding module on substrate degradation by the human chitotriosidase

Human chitotriosidase (HCHT) is one of two active glycoside hydrolase family 18 chitinases produced by humans. The enzyme is associated with several diseases and is thought to play a role in the anti-parasite responses of the innate immune system. HCHT occurs in two isoforms, one 50kDa (HCHT50) and one 39kDa variant (HCHT39). Common for both isoforms is a catalytic domain with the (β/α)8 TIM barrel fold. HCHT50 has an additional linker-region, followed by a C-terminal carbohydrate-binding module (CBM) classified as CBM family 14 in the CAZy database. To gain further insight into enzyme functionality and especially the effect of the CBM, we expressed both isoforms and compared their catalytic properties on chitin and high molecular weight chitosans. HCHT50 degrades chitin faster than HCHT39 and much more efficiently. Interestingly, both HCHT50 and HCHT39 show biphasic kinetics on chitosan degradation where HCHT50 is faster initially and HCHT39 is faster in the second phase. Moreover, HCHT50 produces distinctly different oligomer distributions than HCHT39. This is likely due to increased transglycosylation activity for HCHT50 due the CBM extending the positive subsites binding surface and therefore promoting transglycosylation. Finally, studies with both chitin and chitosan showed that both isoforms have a similarly low degree of processivity. Combining functional and structural features of the two isoforms, it seems that HCHT combines features of exo-processive and endo-nonprocessive chitinases with the somewhat unusual CBM14 to reach a high degree of efficiency, in line with its alleged physiological task of being a “complete” chitinolytic machinery by itself.

Heterologous expression and characterization of detergent stable endoglucanase EG5B from Paenibacillus sp. IHB B 3084

The endoglucanase gene EG5B of 1611bp with a predicted molecular weight of 58.6kDa from Paenibacillus sp. IHB B 3084 was cloned and expressed in Escherichia coli BL21(DE3). The analysis of deduced amino acid sequence revealed a modular structure of the endoglucanase EG5B with an N-terminal catalytic domain of glycosyl hydrolase family 5 and a C-terminal carbohydrate-binding module of family 3. The purified enzyme showed high hydrolytic activity on carboxymethylcellulose, low activity on p-nitrophenyl β-d-cellobioside, avicel and filter paper, and no activity on microcrystalline cellulose, p-nitrophenyl β-d-glucoside, cellobiose and salicin as substrates. The enzyme was mild-alkaline active with optimum activity at pH 7–8 and stable over broad pH range. The temperature optimum was at 50°C with >50% activity over 30–60°C. The enzyme stability with >63% residual activity towards non-ionic and anionic surfactants and commercial detergents suggested its compatibility as an additive to detergents.

PROTEIN TARGETING TO STARCH is required for localising GRANULE-BOUND STARCH SYNTHASE to starch granules and for normal amylose synthesis in Arabidopsis.

The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.

A novel family 19 chitinase from the marine-derived Pseudoalteromonas tunicata CCUG 44952T: Heterologous expression, characterization and antifungal activity

The Ptchi19 gene of the marine Pseudoalteromonas tunicata CCUG 44952T was cloned and expressed in Escherichia coli. The recombinant chitinase PtChi19p of 483 amino acids has a molecular weight of 53.5kDa and a multi-domain structure characteristic of family 19 chitinases. The relevant constituents of this multi-domain structure are the domain (D132–A155) where the active site is located, and the domain (A437–W479) that includes a C-terminal carbohydrate-binding module 5. The purified protein was active in the temperature range of 20–50°C and at pH values of 6–9.5, maintaining a high stability under suboptimal conditions and in the presence of different metal ions. The recombinant enzyme hydrolyzed colloidal and crystalline chitin, as well as p-NP N-acetyl-β-d-glucosaminide. As PtChi19p exhibited antifungal activity against phytopathogenic and human pathogenic fungi, it could be used as an alternative biofungicide.

Biocatalytic properties and substrate-binding ability of a modular GH10 β-1,4-xylanase from an insect-symbiotic bacterium, Streptomyces mexicanus HY-14.

The gene (1350-bp) encoding a modular β-1,4-xylanase (XylU), which consists of an N-terminal catalytic GH10 domain and a C-terminal carbohydrate-binding module 2 (CBM 2), from Streptomyces mexicanus HY-14 was cloned and functionally characterized. The purified His-tagged recombinant enzyme (rXylU, 44.0 kDa) was capable of efficiently hydrolyze diverse xylosidic compounds, p-nitrophenyl-cellobioside, and p-nitrophenyl-xylopyranoside when incubated at pH 5.5 and 65°C. Especially, the specific activities (649.8 U/mg and 587.0 U/mg, respectively) of rXylU toward oat spelts xylan and beechwood xylan were relatively higher than those (<500.0 U/mg) of many other GH10 homologs toward the same substrates. The results of enzymatic degradation of birchwood xylan and xylooligosaccharides (xylotriose to xylohexaose) revealed that rXylU preferentially hydrolyzed the substrates to xylobiose (>75%) as the primary degradation product. Moreover, a small amount (4%<) of xylose was detected as the degradation product of the evaluated xylosidic substrates, indicating that rXylU was a peculiar GH10 β-1,4-xylanase with substrate specificity, which was different from its retaining homologs. A significant reduction of the binding ability of rXylU caused by deletion of the C-terminal CBM 2 to various insoluble substrates strongly suggested that the additional domain might considerably contribute to the enzyme-substrate interaction.

Crystallization and preliminary X-ray diffraction analysis of Hypocrea jecorina Cel7A in two new crystal forms.

Cel7A (previously known as cellobiohydrolase I) from Hypocrea jecorina was crystallized in two crystalline forms, neither of which have been previously reported. Both forms co-crystallize under the same crystallization conditions. The first crystal form belonged to space group C2, with unit-cell parameters a=152.5, b=44.9, c=57.6 Å, β=101.2°, and diffracted X-rays to 1.5 Å resolution. The second crystal form belonged to space group P6₃22, with unit-cell parameters a=b≃155, c≃138 Å, and diffracted X-rays to 2.5 Å resolution. The crystals were obtained using full-length Cel7A, which consists of a large 434-residue N-terminal catalytic domain capable of cleaving cellulose, a 27-residue flexible linker and a small 36-residue C-terminal carbohydrate-binding module (CBM). However, a preliminary analysis of the electron-density maps suggests that the linker and CBM are disordered in both crystal forms. Complete refinement and structure analysis are currently in progress.

FLOURY ENDOSPERM6 encodes a CBM48 domain‐containing protein involved in compound granule formation and starch synthesis in rice endosperm

Starch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and related factors have been identified for starch biosynthesis, unknown players remain to be identified, given that it is a complicated and sophisticated process. The endosperm of rice (Oryza sativa) has been used for the study of starch synthesis. Here, we report the cloning and characterization of the FLOURY ENDOSPERM6 (FLO6) gene in rice. In the flo6 mutant, the starch content is decreased and the normal physicochemical features of starch are changed. Significantly, flo6 mutant endosperm cells show obvious defects in compound granule formation. Map-based cloning showed that FLO6 encodes a protein of unknown function. It harbors an N-terminal transit peptide that ensures its correct localization and functions in the plastid, and a C-terminal carbohydrate-binding module 48 (CBM48) domain that binds to starch. Furthermore, FLO6 can interact with isoamylase1 (ISA1) both in vitro and in vivo, whereas ISA1 does not bind to starch directly. We thus propose that FLO6 may act as a starch-binding protein involved in starch synthesis and compound granule formation through a direct interaction with ISA1 in developing rice seeds. Our data provide a novel insight into the role of proteins with the CBM48 domain in plant species.

Structural and Functional Characterization of Pseudomonas aeruginosa AlgX: ROLE OF AlgX IN ALGINATE ACETYLATION

The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition "pinch point" that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.