Genome-wide characterization of the cellulose synthase gene superfamily in Pyrus bretschneideri and reveal its potential role in stone cell formation.

Members of the cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily participate in cellulose and hemicellulose synthesis in the plasma membrane. The members of this superfamily are vital for cell wall construction during plant growth and development. However, little is known about their function in pear fruit, a model for Rosaceae species and for fleshy fruit development. In our research, a total of 36 CesA/Csl family members were identified from the pear and were grouped into six subfamilies (CesA, CslB, CslC, CslD, CslE, and CslG) according to phylogenetic relationships. We performed a protein sequence physicochemical analysis, phylogenetic tree construction, a gene structure, a conserved domain, and chromosomal localization analysis. The results indicated that most of the CesA/Csl genes from pear are closely related to genes in Arabidopsis, but these families have unique characteristics in terms of their gene structure, chromosomal localization, phylogeny, and deduced protein sequences, suggesting that they have evolved through different processes. Tissue expression analysis results showed that most of the CesA/Csl genes were constitutively expressed at different levels in different organs. Furthermore, the expression levels of four genes (Pbr032894.2, Pbr016107.1, Pbr00518.1, and Pbr034218.1) tended to first increase and then decrease during fruit development, implying that these four genes may be involved in the development of stone cells of pear fruit. Our results may help elucidate the evolutionary history and functional differences of the CesA/Csl genes in pear and lay a foundation for further investigation of the CesA/Csl genes in pear and other Rosaceae species.

Comparative study on four amylosucrases from Bifidobacterium species.

Amylosucrase (ASase) is α-glucan-producing enzyme. Four putative ASase genes (bdas, blas, bpas, and btas) were cloned from Bifidobacterium sp. and expressed in Escherichia coli. All ASases from Bifidobacterium sp. (BAS) displayed typical ASase properties with slightly different characteristics. Among the BASs studied, BdAS and BpAS showed maximal enzyme activities at 35 and 30 °C, respectively, whereas BlAS and BtAS were maximally active at higher temperatures, i.e., 45 and 50 °C, respectively. BpAS exhibited optimum pH under slightly basic conditions (pH 8.0), while BdAS, BlAS, and BtAS preferred weakly acidic conditions (pH 5.0-6.0). All BASs showed higher isomerization activities. Particularly, BlAS produced more trehalulose than turanose. Although polymerization was the highest for BtAS, BtAS synthesized α-1, 4-glucans with a lower degree of polymerization than that of the other BASs. The versatile properties of the BASs described could contribute to the efficient production of highly valuable biomaterials for the agriculture, food, and pharmaceutical industries.

Molecular identification of a cyclodextrin glycosyltransferase-producing microorganism and phylogenetic assessment of enzymatic activities.

Cyclodextrin glycosyltransferases (CGTases) are important enzymes in the biotechnology field because they catalyze starch conversion into cyclodextrins and linear oligosaccharides, which are used in food, pharmaceutical and cosmetic industries. The CGTases are classified according to their product specificity in α-, ß-, α/ß- and γ-CGTases. As molecular markers are the preferred tool for bacterial identification, we employed six molecular markers (16S rRNA, dnaK, gyrB, recA, rpoB and tufA) to test the identification of a CGTase-producing bacterial strain (DF 9R) in a phylogenetic context. In addition, we assessed the phylogenetic relationship of CGTases along bacterial evolution. The results obtained here allowed us to identify the strain DF 9R as Paenibacillus barengoltzii, and to unveil a complex origin for CGTase types during archaeal and bacterial evolution. We postulate that the α-CGTase activity represents the ancestral type, and that the γ-activity may have derived from ß-CGTases.

Overexpression of a glycogenin, CmGLG2, enhances floridean starch accumulation in the red alga Cyanidioschyzon merolae.

Microalgae accumulate energy-reserved molecules, such as triacylglycerol and carbohydrates, which are suitable feedstocks for renewable energies such as biodiesel and bioethanol. However, the molecular mechanisms behind the microalgae accumulating these molecules require further elucidation. Recently, we have reported that the target of rapamycin (TOR)-signaling is a major pathway to regulate floridean starch synthesis by changing the phosphorylation status of CmGLG1, a glycogenin generally required for the initiation of starch/glycogen synthesis, in the unicellular red alga Cyanidioschyzon merolae. In the present study, we confirmed that another glycogenin, CmGLG2, is also involved in the floridean starch synthesis in this alga, since the CmGLG2 overexpression resulted in a two-fold higher floridean starch content in the cell. The results indicate that both glycogenin isoforms play an important role in floridean starch synthesis in C. merolae, and would be a potential target for improvement of floridean starch production in microalgae.

Single-nucleotide polymorphisms(SNPs) in a sucrose synthase gene are associated with wood properties in Catalpa fargesii bur.

BACKGROUND: Association study is a powerful means for identifying molecular markers, such as single-nucleotide polymorphisms (SNPs) associated with important traits in forest trees. Catalpa fargesii Bur is a valuable commercial tree in China and identifying SNPs that associate with wood property would make a foundation of the marker-assisted breeding in the future. However, related work has not been reported yet. RESULTS: We cloned a 2887 bp long sucrose synthase (SUS) gene from the genome of C. fargesii, which is a key enzyme in sucrose metabolism and also associated to wood formation in trees, coding 806 amino acids that expressed mainly in young branches, xylem, and leaves according to real-time quantitative PCR. Then we identified allelic variations of CfSUS associated with nine wood quality associated traits in Catalpa fargesii Bur. Totally, 135 SNPs were identified through cloning and sequencing the CfSUS locus from a mapping population (including 93 unrelated individuals) and 47 of which were genotyped as common SNPs (minor allele frequency > 5%) in the association population that comprised of 125 unrelated individuals collected from main distribution area. Nucleotide diversity and linkage disequilibrium (LD) analysis showed CfSUS has a relative low SNP diversity (πT = 0.0034) and low LD (r2 dropped below 0.1 within 1600 bp). Using the association analysis, we found 11 common SNPs and 14 haplotypes were significantly associated with the traits (false discovery rate, Q<0.1), explaining 3.21-12.41% of the phenotypic variance. These results provide molecular markers above associated with wood basic density, pore rate, and six other traits of wood, which have potential applications in breeding of Catalpa fargesii Bur. CONCLUSION: We first cloned a SUS gene in C. fargesii, then identified several SNPs and haplotypes that associated with wood properties within this gene, suggesting CfSUS participates in the wood formation of C. fargesii. Moreover, molecular markers we identified in this study may be applied into marker-assisted breeding of C. fargesii in the future.

Effect of 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene on cellulose synthase A (CesA) genes in flax (Linum usitatissimum L. 'Nike') seedlings.

INTRODUCTION: Cellulose microfibril is a major cell wall polymer that plays an important role in the growth and development of plants. The gene cellulose synthase A (CesA), encoding cellulose synthases, is involved in the synthesis of cellulose microfibrils. However, the regulatory mechanism of CesA gene expression is not well understood, especially during the early developmental stages. OBJECTIVE: To identify factor(s) that regulate the expression of CesA genes and ultimately control seedling growth and development. METHODS: The presence of cis-elements in the promoter region of the eight CesA genes identified in flax (Linum usitatissimum L. 'Nike') seedlings was verified, and three kinds of ethylene-responsive cis-elements were identified in the promoters. Therefore, the effect of ethylene on the expression of four selected CesA genes classified into Clades 1 and 6 after treatment with 10-4 and 10-3 M 1-aminocyclopropane-1-carboxylic acid (ACC) was examined in the hypocotyl of 4-6-day-old flax seedlings. RESULTS: ACC-induced ethylene either up- or down-regulated the expression of the CesA genes depending on the clade to which these genes belonged, age of seedlings, part of the hypocotyl, and concentration of ACC. CONCLUSION: Ethylene might be one of the factors regulating the expression of CesA genes in flax seedlings.

Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12.

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and ß-d-glucose-1-phosphate ( kcat = 1.1 s-1, Km = 1.05 mM, and kcat/ Km = 1.12 × 103 M-1 s-1). Additionally, it was also shown that in the presence of ß-d-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-d-glucitol, l-sorbose, d-sorbitol, or l-iditol as a substitute for d-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a ß-phosphoglucomutase that catalyzes the isomerization of ß-d-glucose-1-phosphate ( kcat = 21 s-1, Km = 18 µM, and kcat/ Km = 1.1 × 106 M-1 s-1) to d-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with ß-d-allose-1-phosphate, ß-d-mannose-1-phosphate, and ß-d-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of α-(1,2)-d-glucose-d-glycerate with a kcat = 2.1 s-1, Km = 69 µM, and kcat/ Km = 3.1 × 104 M-1 s-1.

Cellulose synthase 'class specific regions' are intrinsically disordered and functionally undifferentiated.

Cellulose synthases (CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls. Seed plant CESA isoforms cluster in six phylogenetic clades, whose non-interchangeable members play distinct roles within cellulose synthesis complexes (CSCs). A 'class specific region' (CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms. We investigated CESA isoform specificity in the moss, Physcomitrella patens (Hedw.) B. S. G. to gain evolutionary insights into CESA structure/function relationships. Like seed plants, P. patens has oligomeric rosette-type CSCs, but the PpCESAs diverged independently and form a separate CESA clade. We showed that P. patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore-negative phenotype of a ppcesa5 knockout line. Thus, non-interchangeable CESA classes evolved separately in mosses and seed plants. However, testing of chimeric moss CESA genes for complementation demonstrated that functional class-specificity is not determined by the CSR. Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class-specific sequences without selection for class-specific function.

Formation of wood secondary cell wall may involve two type cellulose synthase complexes in Populus.

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.

Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation.

The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process.

Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean (Glycine max).

C-Glucosyltransferase is an enzyme that mediates carbon-carbon bond formation to generate C-glucoside metabolites. Although it has been identified in several plant species, the catalytic amino acid residues required for C-glucosylation activity remain obscure. Here, we identified a 2-hydroxyflavanone C-glucosyltransferase (UGT708D1) in soybean. We found that three residues, His20, Asp85, and Arg292, of UGT708D1 were located at the predicted active site and evolutionarily conserved. The substitution of Asp85 or Arg292 with alanine destroyed C-glucosyltransferase activity, whereas the substitution of His20 with alanine abolished C-glucosyltransferase activity but enabled O-glucosyltransferase activity. The catalytic mechanism is discussed on the basis of the findings.

Overexpression of poplar xylem sucrose synthase in tobacco leads to a thickened cell wall and increased height.

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.

Expression divergence of cellulose synthase (CesA) genes after a recent whole genome duplication event in Populus.

MAIN CONCLUSION: Secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules. Gene duplication is an important mechanism for functional divergence of genes. Secondary cell wall-associated cellulose synthase genes (CesA4, CesA7 and CesA8) are duplicated in Populus plants due to a recent whole genome duplication event. Here, we demonstrate that duplicate CesA genes show tissue-dependent expression divergence in Populus plants. Real-time PCR analysis of Populus CesA genes suggested that Pt × tCesA8-B was more highly expressed than Pt × tCesA8-A in phloem and secondary xylem tissue of mature stem. Histochemical and histological analyses of transformants expressing a GFP-GUS fusion gene driven by Populus CesA promoters revealed that the duplicate CesA genes showed different expression patterns in phloem fibers, secondary xylem, root cap and leaf trichomes. We predicted putative cis-regulatory motifs that regulate expression of secondary cell wall-associated CesA genes, and identified 19 motifs that are highly conserved in the CesA gene family of eudicotyledonous plants. Furthermore, a transient transactivation assay identified candidate transcription factors that affect levels and patterns of expression of Populus CesA genes. The present study reveals that secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules.

Genome-wide identification and expression profile analysis of citrus sucrose synthase genes: investigation of possible roles in the regulation of sugar accumulation.

Sucrose synthase (Sus) (EC 2.4.1.13) is a key enzyme for the sugar accumulation that is critical to form fruit quality. In this study, extensive data-mining and PCR amplification confirmed that there are at least six Sus genes (CitSus1-6) in the citrus genome. Gene structure and phylogeny analysis showed an evolutionary consistency with other plant species. The six Sus genes contain 12-15 exons and 11-14 introns and were evenly distributed into the three plant Sus groups (CitSus1 and CitSus2 in the Sus I group, CitSus3 and CitSus6 in the Sus II group, and CitSus4 and CitSus5 in the Sus III group). Transcripts of these six CitSus genes were subsequently examined. For tissues and organs, CitSus1 and 2 were predominantly expressed in fruit juice sacs (JS) whereas CitSus3 and 4 were predominantly expressed in early leaves (immature leaves), and CitSus5 and 6 were predominantly expressed in fruit JS and in mature leaves. During fruit development, CitSus5 transcript increased significantly and CitSus6 transcript decreased significantly in fruit JS. In the fruit segment membrane (SM), the transcript levels of CitSus2 and 5 were markedly higher and the abundant levels of CitSus3 and 6 gradually decreased. Moreover, transcript levels of CitSus1-4 examined were higher and the CitSus5 transcript level was lower in the fruit SM than in fruit JS, while CitSus6 had a similar transcript level in fruit JS and SM. In addition, transcripts of CitSus1-6 responded differently to dehydration in mature leaves or to mild drought stress in fruit JS and SM. Finally, the possible roles of Sus genes in the regulation of sugar accumulation are discussed; however, further study is required.

Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

Bioactive gibberellins (GAs) comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

Allelic variation in a cellulose synthase gene (PtoCesA4) associated with growth and wood properties in Populus tomentosa.

Lignocellulosic biomass from trees provides a renewable feedstock for biofuels, lumber, pulp, paper, and other uses. Dissecting the mechanism underlying natural variation of the complex traits controlling growth and lignocellulose biosynthesis in trees can enable marker-assisted breeding to improve wood quality and yield. Here, we combined linkage disequilibrium (LD)-based association analysis with traditional linkage analysis to detect the genetic effect of a Populus tomentosa cellulose synthase gene, PtoCesA4. PtoCesA4 is strongly expressed in developing xylem and leaves. Nucleotide diversity and LD in PtoCesA4, sampled from the P. tomentosa natural distribution, revealed that PtoCesA4 harbors high single nucleotide polymorphism (SNP) diversity (πT = 0.0080 and θw = 0.0098) and low LD (r(2) ≥ 0.1, within 1400 bp), demonstrating that the potential of a candidate-gene-based LD approach in understanding the molecular basis underlying quantitative variation in this species. By combining single SNP, multi-SNP, and haplotype-based associations in an association population of 460 individuals with single SNP linkage analysis in a family-based linkage populations (1200 individuals), we identified three strong associations (false discovery rate Q < 0.05) in both populations. These include two nonsynonymous markers (SNP49 associated with α-cellulose content and SNP59 associated with fiber width) and a noncoding marker (SNP18 associated with α-cellulose content). Variation in RNA transcript abundance among genotypic classes of SNP49 was confirmed in these two populations. Therefore, combining different methods allowed us to examine functional PtoCesA4 allelic variation underlying natural variation in complex quantitative traits related to growth and lignocellulosic biosynthesis.

Infection structure-specific expression of ß-1,3-glucan synthase is essential for pathogenicity of Colletotrichum graminicola and evasion of ß-glucan-triggered immunity in maize.

ß-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of ß-1,3-glucan is unknown. We functionally characterized the ß-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive ß-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and ß-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of ß-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of ß-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while ß-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading ß-glucan-triggered immunity.

Characterization of Ruminococcus albus cellodextrin phosphorylase and identification of a key phenylalanine residue for acceptor specificity and affinity to the phosphate group.

Ruminococcus albus has the ability to intracellularly degrade cello-oligosaccharides primarily via phosphorolysis. In this study, the enzymatic characteristics of R. albus cellodextrin phosphorylase (RaCDP), which is a member of glycoside hydrolase family 94, was investigated. RaCDP catalyzes the phosphorolysis of cellotriose through an ordered 'bi bi' mechanism in which cellotriose binds to RaCDP before inorganic phosphate, and then cellobiose and glucose 1-phosphate (Glc1P) are released in that order. Among the cello-oligosaccharides tested, RaCDP had the highest phosphorolytic and synthetic activities towards cellohexaose and cellopentaose, respectively. RaCDP successively transferred glucosyl residues from Glc1P to the growing cello-oligosaccharide chain, and insoluble cello-oligosaccharides comprising a mean of eight residues were produced. Sophorose, laminaribiose, ß-1,4-xylobiose, ß-1,4-mannobiose and cellobiitol served as acceptors for RaCDP. RaCDP had very low affinity for phosphate groups in both the phosphorolysis and synthesis directions. A sequence comparison revealed that RaCDP has Gln at position 646 where His is normally conserved in the phosphate binding sites of related enzymes. A Q646H mutant showed approximately twofold lower apparent K(m) values for inorganic phosphate and Glc1P than the wild-type. RaCDP has Phe at position 633 corresponding to Tyr and Val in the +1 subsites of cellobiose phosphorylase and N,N'-diacetylchitobiose phosphorylase, respectively. A F633Y mutant showed higher preference for cellobiose over ß-1,4-mannobiose as an acceptor substrate in the synthetic reaction than the wild-type. Furthermore, the F633Y mutant showed 75- and 1100-fold lower apparent Km values for inorganic phosphate and Glc1P, respectively, in phosphorolysis and synthesis of cellotriose.

Consensus engineering of sucrose phosphorylase: the outcome reflects the sequence input.

Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tried only a few times mainly because of the problems determining the consensus in highly variable regions. We show that this problem can be solved by replacing such problematic regions by the corresponding sequence of the natural homologue closest to the consensus. When one or a few sub-families are overrepresented in the MSA the consensus sequence is a biased representation of the sequence space. We examine the influence of this bias by constructing three consensus sequences using different MSAs of sucrose phosphorylase (SP). Each consensus enzyme contained about 70 mutations compared to its closest natural homologue and folded correctly and displayed activity on sucrose. Correlation analysis revealed that the family's co-evolution network was kept intact, which is one of the main advantages of full-length consensus design. The consensus enzymes displayed an "average" thermostability, that is, one that is higher than some but not all known representatives. We cautiously present practical rules for the design of consensus sequences, but warn that the measure of success depends on which natural enzyme is used as point of comparison.

Four novel cellulose synthase (CESA) genes from Birch (Betula platyphylla Suk.) involved in primary and secondary cell Wall biosynthesis.

Cellulose synthase (CESA), which is an essential catalyst for the generation of plant cell wall biomass, is mainly encoded by the CesA gene family that contains ten or more members. In this study; four full-length cDNAs encoding CESA were isolated from Betula platyphylla Suk., which is an important timber species, using RT-PCR combined with the RACE method and were named as BplCesA3, -4, -7 and -8. These deduced CESAs contained the same typical domains and regions as their Arabidopsis homologs. The cDNA lengths differed among these four genes, as did the locations of the various protein domains inferred from the deduced amino acid sequences, which shared amino acid sequence identities ranging from only 63.8% to 70.5%. Real-time RT-PCR showed that all four BplCesAs were expressed at different levels in diverse tissues. Results indicated that BplCESA8 might be involved in secondary cell wall biosynthesis and floral development. BplCESA3 appeared in a unique expression pattern and was possibly involved in primary cell wall biosynthesis and seed development; it might also be related to the homogalacturonan synthesis. BplCESA7 and BplCESA4 may be related to the formation of a cellulose synthase complex and participate mainly in secondary cell wall biosynthesis. The extremely low expression abundance of the four BplCESAs in mature pollen suggested very little involvement of them in mature pollen formation in Betula. The distinct expression pattern of the four BplCesAs suggested they might participate in developments of various tissues and that they are possibly controlled by distinct mechanisms in Betula.