The Arabidopsis Cellulose Synthase Complex: A Proposed Hexamer of CESA Trimers in an Equimolar Stoichiometry

Cellulose is the most abundant renewable polymer on Earth and a major component of the plant cell wall. In vascular plants, cellulose synthesis is catalyzed by a large, plasma membrane-localized cellulose synthase complex (CSC), visualized as a hexameric rosette structure. Three unique cellulose synthase (CESA) isoforms are required for CSC assembly and function. However, elucidation of either the number or stoichiometry of CESAs within the CSC has remained elusive. In this study, we show a 1:1:1 stoichiometry between the three Arabidopsis thaliana secondary cell wall isozymes: CESA4, CESA7, and CESA8. This ratio was determined utilizing a simple but elegant method of quantitative immunoblotting using isoform-specific antibodies and ³⁵S-labeled protein standards for each CESA. Additionally, the observed equimolar stoichiometry was found to be fixed along the axis of the stem, which represents a developmental gradient. Our results complement recent spectroscopic analyses pointing toward an 18-chain cellulose microfibril. Taken together, we propose that the CSC is composed of a hexamer of catalytically active CESA trimers, with each CESA in equimolar amounts. This finding is a crucial advance in understanding how CESAs integrate to form higher order complexes, which is a key determinate of cellulose microfibril and cell wall properties.     

Extraction of natural products from bark of <Emphasis Type="Italic">Betula pendula</Emphasis> using ionic liquids

Introduction

Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.

Objectives

To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.

Methods

The extracted compounds were analyzed by LC/MS and GC/MS.

Results

The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.

Conclusion

From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.

     

Adhesion of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> biofilms to glass,stainless steel and cellulose

Objectives

The adhesion of colloidal probes of stainless steel, glass and cellulose to Pseudomonas fluorescens biofilms was examined using atomic force microscopy (AFM) to allow comparisons between surfaces to which biofilms might adhere.

Results

Biofilm was grown on a stainless steel substrate and covered most of the surface after 96 h. AFM approach and retraction curves were obtained when the biofilm was immersed in a tryptone/soy medium. On approach, all the colloidal probes experienced a long non-contact phase more than 100 nm in length, possibly due to the steric repulsion by extracellular polymers from the biofilm and hydrophobic effects. Retraction data showed that the adhesion varied from position to position on the biofilm. The mean value of adhesion of glass to the biofilm (48 ± 7 nN) was the greatest, followed by stainless steel (30 ± 7 nN) and cellulose (7.8 ± 0.4 nN).

Conclusion

The method allows understanding of adhesion between the three materials and biofilm, and development of a better strategy to remove the biofilm from these surfaces relevant to different industrial applications.

     

Functional analysis of complexes with mixed primary and secondary cellulose synthases

In higher plants, cellulose is synthesized by cellulose synthase complexes, which contain multiple isoforms of cellulose synthases (CESAs). Among the total 10 CESA genes in Arabidopsis, recessive mutations at three of them cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis (irx1^cesa8, irx3^cesa7 and irx5^cesa4). These CESA genes are considered secondary cell wall CESAs. The others (the function CESA10 is still unknown) are thought to be specialized for cellulose synthesis in the primary cell wall. A split-ubiquitin membrane yeast two-hybrid system was used to assess interactions among four primary CESAs (CESA1, CESA2, CESA3, CESA6) and three secondary CESAs (CESA4, CESA7, CESA8). Our results showed that primary CESAs could physically interact with secondary CESAs in a limited fashion. Analysis of transgenic lines showed that CESA1 could partially rescue irx1^cesa8 null mutants, resulting in complementation of the plant growth defect, collapsed xylem and cellulose content deficiency. These results suggest that mixed primary and secondary CESA complexes are functional using experimental set-ups.     

The effect of <Emphasis Type="Italic">INDEHISCENT</Emphasis> point mutations on silique shatter resistance in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>)

Key message

This study elucidates the influence of indehiscent mutations on rapeseed silique shatter resistance. A phenotype with enlarged replum-valve joint area and altered cell dimensions in the dehiscence zone is described.

Abstract

Silique shattering is a major factor reducing the yield stability of oilseed rape (Brassica napus). Attempts to improve shatter resistance often include the use of mutations in target genes identified from Arabidopsis (Arabidopsis thaliana). A variety of phenotyping methods assessing the level of shatter resistance were previously described. However, a comparative and comprehensive evaluation of the methods has not yet been undertaken. We verified the increase of shatter resistance in indehiscent double knock-down mutants obtained by TILLING with a systematic approach comparing three independent phenotyping methods. A positive correlation of silique length and shatter resistance was observed and accounted for in the analyses. Microscopic studies ruled out the influence of different lignification patterns. Instead, we propose a model to explain increased shattering resistance of indehiscent rapeseed mutants by altered cell shapes and sizes within the contact surfaces of replum and valves.

     

Design,molecular docking,synthesis, characterization,biological activity evaluation (against MES model), <Emphasis Type="Italic">in-silico</Emphasis> biological activity spectrum (PASS analysis), toxicological and predicted oral rat LD<Subscript>50</Subscript> studies of novel sulphonamide derivatives

Background

Among the reported potential agents to treat the epilepsy, sulphonamides are important and their significance cannot be ignored. A series of substituted 4-amino-benzene sulfonamides were designed, keeping in view the structural requirement of pharmacophore.

Methods

Lipinski rule of five has been calculated; failure to Lipinski rule was not observed. Docking was performed through AutoDock Vina. Molecules have been screened out through docking. Compounds were synthesized and characterized through IR, ¹HNMR, ¹³C NMR, Mass and elemental analysis. The anticonvulsant activity of the synthesized compounds was assessed using the Maximal Electroshock Seizure (MES) model. In-silico biological activity spectrum, toxicological studies, predicted oral rats LD50 were performed.

Results

Docking studies showed good interaction with lyase (Oxo-acid) - human carbonic anhydrase-I (1AZM). The in-silico studies proved them to be with good drug-likeness properties, especially 4-(3-Acetyl-phenylamino)-methyl)-benzenesulfonamide (2g). These results revealed that the synthesized compounds (1a-1c, 2a-2q) exhibited promising anticonvulsant effect against MES model for inhibition of Lyase- Human Carbonic Anhydrase-I.

Conclusion

After investigating all the results, the compound 4-(3-Acetyl-phenylamino)-methyl)-benzenesulfonamide (2g) is found to be best in the series. A comparatively good activity of compound 2g suggests us that sulphonamide can be leads to further optimization for building potent and chemically diversified anti-convulsant agents.

     

Expansins: roles in plant growth and potential applications in crop improvement

Key message

Results from various expansin related studies have demonstrated that expansins present an opportunity to improve various crops in many different aspects ranging from yield and fruit ripening to improved stress tolerance.

Abstract

The recent advances in expansin studies were reviewed. Besides producing the strength that is needed by the plants, cell walls define cell shape, cell size and cell function. Expansins are cell wall proteins which consist of four sub families; α-expansin, β-expansin, expansin-like A and expansin-like B. These proteins mediate cell wall loosening and they are present in all plants and in some microbial organisms and other organisms like snails. Decades after their initial discovery in cucumber, it is now clear that these small proteins have diverse biological roles in plants. Through their ability to enable the local sliding of wall polymers by reducing adhesion between adjacent wall polysaccharides and the part they play in cell wall remodeling after cytokinesis, it is now clear that expansins are required in almost all plant physiological development aspects from germination to fruiting. This is shown by the various reports from different studies using various molecular biology approaches such as gene achieve these many roles through their non-enzymatic wall loosening ability. This paper reviews and summarizes some of the reported functions of expansins and outlines the potential uses of expansins in crop improvement programs.

     

Biomass digestibility is predominantly affected by three factors of wall polymer features distinctive in wheat accessions and rice mutants

Background

Wheat and rice are important food crops with enormous biomass residues for biofuels. However, lignocellulosic recalcitrance becomes a crucial factor on biomass process. Plant cell walls greatly determine biomass recalcitrance, thus it is essential to identify their key factors on lignocellulose saccharification. Despite it has been reported about cell wall factors on biomass digestions, little is known in wheat and rice. In this study, we analyzed nine typical pairs of wheat and rice samples that exhibited distinct cell wall compositions, and identified three major factors of wall polymer features that affected biomass digestibility.

Results

Based on cell wall compositions, ten wheat accessions and three rice mutants were classified into three distinct groups each with three typical pairs. In terms of group I that displayed single wall polymer alternations in wheat, we found that three wall polymer levels (cellulose, hemicelluloses and lignin) each had a negative effect on biomass digestibility at similar rates under pretreatments of NaOH and H₂SO₄ with three concentrations. However, analysis of six pairs of wheat and rice samples in groups II and III that each exhibited a similar cell wall composition, indicated that three wall polymer levels were not the major factors on biomass saccharification. Furthermore, in-depth detection of the wall polymer features distinctive in rice mutants, demonstrated that biomass digestibility was remarkably affected either negatively by cellulose crystallinity (CrI) of raw biomass materials, or positively by both Ara substitution degree of non-KOH-extractable hemicelluloses (reverse Xyl/Ara) and p-coumaryl alcohol relative proportion of KOH-extractable lignin (H/G). Correlation analysis indicated that Ara substitution degree and H/G ratio negatively affected cellulose crystallinity for high biomass enzymatic digestion. It was also suggested to determine whether Ara and H monomer have an interlinking with cellulose chains in the future.

Conclusions

Using nine typical pairs of wheat and rice samples having distinct cell wall compositions and wide biomass saccharification, Ara substitution degree and monolignin H proportion have been revealed to be the dominant factors positively determining biomass digestibility upon various chemical pretreatments. The results demonstrated the potential of genetic modification of plant cell walls for high biomass saccharification in bioenergy crops.

     

A model of hematopoietic bone marrow apoptosis during growth factor deprivation in combination with a cytokine

Background

The process by which blood cells are formed is referred to as hematopoiesis. This process involves a complex sequence of phases that blood cells must complete. During hematopoiesis, a small fraction of cells undergo cell death. Causes of cell death are dependent upon various factors; one such factor being growth factor deprivation.

Methods

In this paper, a mathematical model of hematopoiesis during growth factor deprivation is presented. The model consists of a set of three coupled differential delay equations. Phase plane and linear stability analysis are performed in order to locate and determine stability of fixed points. Numerical simulations of the governing equations are run and provide a visual display of the behavior of the stem cell population undergoing growth factor deprivation. In addition, the effect of cytokine administration is incorporated in the model in an effort to understand how cytokine administration can offset the negative effects of apoptosis caused by growth factor deprivation.

Conclusions

The model produces qualitatively similar results to that observed during serum deprivation. The model captures apoptosis levels of cells at different time points. Additionally, it is shown that cytokine administration stabilizes the stem cell count.

     

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers

A cellulose synthesis complex with a “rosette” shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the “hexamer of trimers” model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.Cellulose, the most abundant biopolymer on Earth, is composed of linear chains of β-1,4 linked d-Glc monomers with repeating structural units of the disaccharide cellobiose. Numerous cellulose polymers cocrystallize to form microfibrils, which provide mechanical strength and rigidity to plants. Its natural abundance makes it an attractive target for many industrial applications, including paper and pulping, construction, and textile manufacture. More recently, cellulose has been used for production of biofuels, such as ethanol (Ragauskas et al., 2006; Langan et al., 2014), and in the form of nanocellulose as a component in advanced composite materials (Reddy et al., 2013; Habibi, 2014). Cellulose microfibrils are synthesized by a large membrane-bound protein complex. In the land plants and charophycean algae, the cellulose synthesis complex (CSC) has a “rosette” shape (Mueller et al., 1976; Mueller and Brown, 1980b; Kimura et al., 1999), and the entire CSC has reported diameters between 24 to 30 nm (Lerouxel et al., 2006). This structural information was revealed by freeze-fracture transmission electron microscopy, showing six lobes in a hexagonal arrangement at the point where the transmembrane helices of multiple cellulose synthase proteins (CESAs) cross the plasma membrane. Recently KORRIGAN, a protein with cellulase activity, has also been implicated as an integral component of the CSC (Vain et al., 2014).Vascular plants produce several different CESA isoforms. For example, Arabidopsis (Arabidopsis thaliana) has 10 different isoforms with 64% to 98% sequence identity (Holland et al., 2000; Richmond, 2000; McFarlane et al., 2014). The different CESA isoforms play specific roles in cellulose synthesis during plant development. In Arabidopsis, CESA1, CESA3, and CESA6 are required for primary cell wall synthesis, while CESA4, CESA7, and CESA8 are required for secondary cell wall synthesis (Gardiner et al., 2003; Taylor et al., 2003; Persson et al., 2007). CESA2, CESA5, and CESA9 play roles in tissue-specific processes and are partially redundant with CESA6, whereas CESA10 is closely related to AtCESA1 but evidently has a minor role in plant development (Somerville, 2006). The absolute number of CESA proteins present in a CSC remains a subject of much speculation, largely because the stoichiometry of the cellulose microfibril remains unresolved (Cosgrove, 2014). The traditional representation of the microfibril has 36 cellulose chains, and based on this, one would expect that each lobe of the rosette CSC contains six CESA proteins responsible for the synthesis of six glucan chains for a total of 36 CESA proteins per rosette CSC (Herth, 1983; Perrin, 2001; Doblin et al., 2002). However, recent studies using different analytical techniques combined with computation report 18 to 24 cellulose chains per microfibril (Fernandes et al., 2011; Thomas et al., 2013; Oehme et al., 2015). A study of cellulose from mung bean (Vigna radiata) primary cell walls, using x-ray diffraction, solid-state NMR, and computational analysis, supports an 18-chain model for a cellulose microfibril (Newman et al., 2013). This implies that the CSC is composed of fewer than 36 CESA proteins or that not all of the proteins in a CSC are simultaneously active. Further, it has been recently reported that the stoichiometry of CESAs 1, 3, and 6 and CESAs 4, 7, and 8 in the primary and secondary cell walls, respectively, is 1:1:1 (Gonneau et al., 2014; Hill et al., 2014). Together, these reports suggest a rosette CSC composed of 18 CESA proteins with three CESAs per lobe as the most likely composition of a rosette CSC to account for an 18-chain cellulose microfibril (Newman et al., 2013; Gonneau et al., 2014; Hill et al., 2014). In addition, it should also be noted that 24 CESA proteins in a rosette CSC with four proteins per lobe is incompatible with a 1:1:1 CESA stoichiometry.Numerous efforts to isolate active CESA proteins directly from plants or by recombinant expression have not been successful, preventing a detailed structural analysis of CESA proteins or the mechanism of plant cellulose synthesis. In contrast, the recently reported crystal structure of cellulose synthase from Rhodoobacter sphaeroides (Morgan et al., 2013) clearly showed that only a single cellulose synthase polypeptide is required for glucan polymerization and also identified the conserved sequence motifs responsible for catalysis. In addition, based on the presence of an 18-residue glucan chain in the protein tunnel, a mechanism for cellulose synthesis and translocation across a cytoplasmic membrane was proposed that also addressed how the alternate d-Glc molecules are inverted during polymer synthesis (Morgan et al., 2013; Omadjela et al., 2013). However, this structure cannot provide insight into the formation of microfibrils from the cellulose chains synthesized by single polypeptides of CESA.The CESA proteins of land plants and their charophycean algal relatives are multidomain single polypeptide chains of approximately 1000 amino acids. They are predicted to have eight transmembrane helices and to have their N- and C-terminal regions facing the cytoplasm (Pear et al., 1996). Although they share sequence similarity with the bacterial counterpart, they also have unique structural features not found in the bacterial enzymes. The N-terminal domain contains a Zn-binding site that may play a role in oligomerization of CESA proteins (Kurek et al., 2002). The putative cytosolic domain, which is flanked by a two-helix N-terminal transmembrane domain and a six-helix C-terminal transmembrane domain (McFarlane et al., 2014; Slabaugh et al., 2014), has D, D, D, QxxRW motifs that are conserved substrate binding and catalytic residues in the glycosyltransferase-2 superfamily (Nagahashi et al., 1995; Pear et al., 1996; Saxena and Brown, 1997; Yoshida et al., 2000). This domain also has a plant-conserved region (P-CR) and a class-specific region (CSR) that are only found in CESAs that form rosette CSCs. Although the roles of these regions are unknown, they are proposed to be involved in regulatory functions, such as interactions with other proteins and oligomerization to form the rosette shape. In the Arabidopsis CESAs, the sequence identity within the P-CR regions is greater than 80%, while in CSR regions, it is only about 40%. A recent computational model of the cytosolic domain of cotton (Gossypium hirsutum) CESA1 provides the first detailed structural model of the catalytic domain of CESA (Sethaphong et al., 2013). This model structure aligns well with the crystal structure of the bacterial cellulose synthase, indicating that a common mechanism exists for cellulose synthesis in bacteria and plants and that CESAs within rosette CSCs contain a single active synthetic site. In addition, this model made it possible to test possible configurations for the assembly of CESA monomers into a functional rosette CSC (Newman et al., 2013; Sethaphong et al., 2013).Our understanding of the mechanism of cellulose biosynthesis in plants at the molecular level is hampered by the lack of an atomic level CESA model. To gain deeper insight into the structure and role of the catalytic domain of CESA in rosette formation, we carried out a structural characterization of the cytosolic domain of Arabidopsis CESA1, a protein that is essential for cellulose synthesis in the primary cell wall (Arioli et al., 1998). The recombinant protein was purified from Escherichia coli in a two-step process that allowed us to obtain low-resolution structural information about the monomeric and trimeric forms of the recombinant protein using small-angle scattering (SAS) techniques. This study provides the first experimental evidence to support the self-assembly of CESAs into a stable trimer complex, revealing the possible role of the catalytic domain in the formation of the rosette CSC. Comparison of the size of the catalytic domain trimer with dimensions of rosette CSCs obtained from TEM studies strongly supports the “hexamer of trimers” model for rosette CSCs. Computational analysis of the scattering data suggested configurations for how the monomers, including the plant-specific P-CR and CSR domains, may be arranged in the trimeric lobes of the rosette CSC. Knowledge of how CESA proteins assemble in the CSC will enable approaches for rational genetic manipulation of plant cell wall synthesis, which offers enormous opportunities to improve feedstocks for the production of sustainable fuels and chemicals.     

High-frequency fire alters soil and plant chemistry but does not lead to nitrogen-limited growth of <Emphasis Type="Italic">Eucalyptus pilularis</Emphasis> seedlings

Background and aims

The current study was undertaken to investigate the mechanism underlying Boron (B)-alleviated phosphate (P) deficiency in Arabidopsis thaliana. Furthermore, we were interested to explore whether this alleviation of P deficiency by B could extend to Brassica crops.

Methods

Arabidopsis thaliana or Brassica oleracea plants were grown under P-sufficient or -deficient condition with or without extra B for 7 days, then shoots and roots of B. oleracea were sampled for analysis of soluble P content while those of A. thaliana were harvested for analysis of total P content, soluble P content and nitric oxide (NO) as well as for cell wall extraction and RNA isolation.

Results

A. thaliana plants showed reduced root growth and decreased P content in the root under P-deficient conditions, but improved root growth when supplemented with additional B. Further analysis revealed that exogenous B elevated the cell wall pectin content and facilitated the release of P in P-deficient seedlings, thus more soluble P was available to sustain growth under P deficiency. Furthermore, B supplement also increased soluble P in P-deficient cabbage (Brassica oleracea var. capitata L.), an economically important vegetable crop. P deficiency alone was sufficient to induce NO accumulation, and in combination with B application further enhanced NO accumulation, while exogenous application of NO scavenger c-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] counteracted this positive effect of B, indicating that NO is positively involved in B-mediated alleviation of P deficiency.

Conclusions

Our study reveals the critical role of B in improving the growth of P-deficient plants, and also provides evidence implicating the involvement of NO signal.

     

Topoisomerase II-associated protein PAT1H1 is involved in the root stem cell niche maintenance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

PAT1H1, one of the homologues of Topoisomerase II-associated protein, is involved in the maintenance of root stem cell niche through the interaction with NINJA.

Abstract

The root stem cell niche, which possesses four mitotically inactive quiescent cells (QC) and the surrounding mitotically active stem cells, is critical for root development in Arabidopsis thaliana. However, the molecular regulation of the maintenance of root stem cell niche identity is still not fully understood. Here we show that one of the homologues of Topoisomerase II-associated protein, here named as PAT1H1, could regulate root stem cell niche identity. The pat1h1 mutant showed higher frequency of QC cell division and root distal stem cell (DSC) differentiation. With a high expression in roots, PAT1H1 was found to interact with the jasmonic acid (JA) signalling negative regulator Novel Interactor of JAZ (NINJA) and thus regulate root DSC niche identity. Consistent with the active QC cell division, which rarely occurs in wild-type controls, the pat1h1 mutant displayed higher expression of CYCB1 in the root stem cell niche. Together our data reveals that PAT1H1 maintains root stem cell niche stability through the interaction with NINJA and the regulation of cell division.

     

Overexpression of phytosulfokine-α induces male sterility and cell growth by regulating cell wall development in Arabidopsis

Key message

Over-production of functional PSK-α in Arabidopsis caused increases in both plant cell growth and biomass and induced male sterility by regulating cell wall development.

Abstract

Phytosulfokine-α (PSK-α) is a novel disulfated pentapeptide hormone that is involved in promoting plant cell growth. Although a role for PSK-α in stimulating protoplast expansion has been suggested, how PSK-α regulates cell growth in planta remains poorly understood. In this study, we found that overexpression of the normal PSK-α precursor gene AtPSK4, which resulted in high levels of PSK-α, caused longer roots and larger leaves with enlarged cells. As expected, these changes were not observed in transgenic plants overexpressing mutated AtPSK4, which generated unsulfated PSK-α. These findings confirmed the role of PSK-α in promoting plant cell growth. Furthermore, we found that overexpressing AtPSK4, but not mutated AtPSK4, induced a phenotype of male sterility that resulted from the failure of fibrous cell wall development in the endothecium. In addition, overexpressing AtPSK4 enhanced expression of a number of genes encoding expansins, which are involved in cell wall loosening. Accordingly, in addition to its role in cell growth, we propose a novel function for PSK-α signaling in the modulation of plant male sterility via regulation of cell wall development.

     

Crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> protein ybgI,a toroidal structure with a dinuclear metal site

Background

The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results

The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Conclusion

The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

     

Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein,monellin, in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with a constitutive GAPDH promoter and modified <Emphasis Type="Italic">N</Emphasis>-terminus

Objectives

To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.

Results

Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.

Conclusions

Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.