The granule-bound starch synthase (GBSSI) gene in the Rosaceae: multiple loci and phylogenetic utility

We sampled the 5' end of the granule-bound starch synthase gene (GBSSI or waxy) in Rosaceae, sequencing 108 clones from 18 species in 14 genera representing all four subfamilies (Amygdaloideae, Maloideae, Rosoideae, and Spiraeoideae), as well as four clones from Rhamnus catharticus (Rhamnaceae). This is the first phylogenetic study to use the 5' portion of this nuclear gene. Parsimony and maximum-likelihood analyses of 941 bases from seven complete and two partial exons demonstrate the presence of two loci (GBSSI-1 and GBSSI-2) in the Rosaceae. Southern hybridization analyses with locus-specific probes confirm that all four Rosaceae subfamilies have at least two GBSSI loci, even though only one locus has been reported in all previously studied diploid flowering plants. Phylogenetic analyses also identify four clades representing four loci in the Maloideae. Phylogenetic relationships inferred from GBSSI sequences are largely compatible with those from chloroplast (cpDNA: ndhF, rbcL) and nuclear ribosomal internal transcribed spacer (nrITS) DNA. Large clades are marked by significant intron variation: a long first intron plus no sixth intron in Maloideae GBSSI-1, a long fourth intron in Rosoideae GBSSI-1, and a GT to GC mutation in the 5' splice site of the fourth intron in all GBSSI-2 sequences. Our data do not support the long-held hypothesis that Maloideae originated from an ancient hybridization between amygdaloid and spiraeoid ancestors. Instead, Spiraeoideae genera (Kageneckia and Vauquelinia) are their closest relatives in all four GBSSI clades.     

Granule-bound starch synthase I. A major enzyme involved in the biogenesis of B-crystallites in starch granules.

Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A- and B-type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule-bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization of the corresponding STA2 locus. This enzyme displays a specific activity an order of magnitude higher than those reported for most vascular plants. This property enables us to report a detailed characterization of amylose synthesis both in vivo and in vitro. We show that GBSSI is capable of synthesizing a significant number of crystalline structures within starch. Quantifications of amount and type of crystals synthesized under these conditions show that GBSSI induces the formation of B-type crystals either in close association with pre-existing amorphous amylopectin or by crystallization of entirely de novo synthesized material.     

Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice

A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a).     

Dolichol-phosphate mannose synthase: structure, function and regulation

Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and dolichol-phosphate-sugars, in which the sugar moiety is mannose or glucose. The sugar transfer from dolichol-phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of dolichol-phosphate-mannose, and particularly on the mammalian enzyme complex involved in the reaction.     

Botulinum and tetanus neurotoxins: structure,function and therapeutic utility

The toxic products of the anaerobic bacteria Clostridium botulinum, Clostridium butyricum, Clostridium barati and Clostridium tetani are the causative agents of botulism and tetanus. The ability of botulinum neurotoxins to disrupt neurotransmission, often for prolonged periods, has been exploited for use in several medical applications and the toxins, as licensed pharmaceutical products, now represent the therapeutics of choice for the treatment for several neuromuscular conditions. Research into the structures and activities of botulinum and tetanus toxins has revealed features of these proteins that might be useful in the design of improved vaccines, effective inhibitors and novel biopharmaceuticals. Here, we discuss the relationships between structure, mechanism of action and therapeutic use.     

Group II introns as phylogenetic tools: structure, function, and evolutionary constraints

Group II introns comprise the majority of noncoding DNA in many plant chloroplast genomes and include the commonly sequenced regions trnK/matK, the rps16 intron, and the rpl16 intron. As demand increases for nucleotide characters at lower taxonomic levels, chloroplast introns may come to provide the bulk of plastome sequence data for assessment of evolutionary relationships in infrageneric, intergeneric, and interfamilial studies. Group II introns have many attractive properties for the molecular systematist: they are confined to organellar genomes in eukaryotes and the majority are single-copy; they share a well-defined and empirically tested secondary and tertiary structure; and many are easily amplified due to highly conserved sequence in flanking exons. However, structure-linked mutation patterns in group II intron sequences are more complex than generally supposed and have important implications for aligning nucleotides, assessing mutational biases in the data, and selecting appropriate models of character evolution for phylogenetic analysis. This paper presents a summary of group II intron function and structure, reviews the link between that structure and specific mutational constraints in group II intron sequences, and discusses strategies for accommodating the resulting complex mutational patterns in subsequent phylogenetic analyses.     

Microbial isopenicillin N synthase genes: structure, function, diversity and evolution.

Clinically and economically, penicillins and cephalosporins are the most important class of the beta-lactam antibiotics. They are produced by a wide variety of microorganisms including numerous species of Streptomyces, some unicellular bacteria and several filamentous fungi. A key step common to their biosynthetic pathways is the conversion of a linear, cysteine-containing tripeptide to a bicyclic beta-lactam antibiotic by isopenicillin N synthase. Recent successes in the cloning and expression of isopenicillin N synthase genes now permit production of a plentiful supply of this enzyme, which may be used for structural and mechanistic studies, or for biotechnological applications in the creation of novel beta-lactam compounds from peptide analogues. New ideas concerning the evolution and prevalence of the penicillin and cephalosporin biosynthetic genes have emerged from studies of isopenicillin N synthase genes.     

Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.     

Correlation between phylogenetic structure and function: examples from deep-sea Shewanella

The genus Shewanella is one of the typical deep-sea bacterial genera. Two isolated deep-sea Shewanella species, Shewanella benthica and Shewanella violacea, were found to be able to grow better under high hydrostatic pressure conditions than at atmospheric pressure. These species are not only piezophilic (barophilic), but also psychrophilic. Many psychrophilic and psychrotolerant Shewanella species have been isolated and characterized from cold environments, such as seawater in Antarctica or the North Sea. Some of these cold-adapted Shewanella were shown to be piezotolerant, meaning that growth occurs in a high-pressure habitat. In this review, we propose that two major sub-genus branches of the genus Shewanella should be recognized taxonomically, one group characterized as high-pressure cold-adapted species that produce substantial amounts of eicosapentaenoic acid, and the other group characterized as mesophilic pressure-sensitive species.     

植物类型III聚酮合酶超家族基因结构、功能及代谢产物

植物聚酮类化合物主要包括酚类、芪类及类黄酮化合物等,在植物花色、防止紫外线伤害、预防病原菌、昆虫危害以及作为植物与环境互作信号分子方面行使着重要的生物学功能。该类化合物具有显著多样的生物学活性,对人体保健及疾病治疗有显著意义。植物类型III 聚酮化合物合酶 (PKS) 在该类化合物生物合成起始反应中行使着关键作用,决定该类化合物基本分子骨架建成和代谢途径碳硫走向,为合成途径关键酶和限速酶。以查尔酮合酶为原型酶的植物类型III PKS超家族是研究系统进化和蛋白结构与功能关系的模式分子家族,目前已经分离得到14种植物类型III PKS基因,这些同祖同源基因及其表达产物既有共性,也表现出许多独特个性,这些个性赋予此类次生代谢产物结构上的多样性。以下综述了植物类型III PKS超家族基因结构、功能及代谢产物研究进展。     

The yeast ATP synthase subunit 4: structure and function

The structure of ATP synthase subunit 4 was determined by using the oligonucleotide probe procedure. This subunit is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its relative molecular mass is 25 kDa. The ATP4 gene was isolated and sequenced. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein 244 amino acids long. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 kDa. Subunit 4 shows homology with the b-subunit of Escherichia coli ATP synthase and the b-subunit of beef heart mitochondrial ATP synthase. By using homologous transformation, a mutant lacking wild subunit 4 was constructed. This mutant is devoid of oxidative phosphorylation and F1 is loosely bound to the membrane. Our data are in favor of a structural relationship between subunit 4 and the mitochondrially-translated subunit 6 during biogenesis of F0.     

Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds

Using genetic variability existing amongst nine pea genotypes (Pisum sativum L.), the biochemical basis of sink strength in developing pea seeds was investigated. Sink strength was considered to be reflected by the rate of starch synthesis (RSS) in the embryo, and sink activity in the seed was reflected by the relative rate of starch synthesis (RRSS). These rates were compared to the activities of three enzymes of the starch biosynthetic pathway [sucrose synthase (Sus), ADP-glucose pyrophosphorylase and starch synthase] at three developmental stages during seed filling (25, 50 and 75% of the dry seed weight). Complete sets of data collected during seed filling for the nine genotypes showed that, for all enzyme activities (expressed on a protein basis), only Sus in the embryo and seed coat was linearly and significantly correlated to RRSS. The contribution of the three enzyme activities to the variability in RSS and RRSS was evaluated by multiple regression analysis for the first two developmental stages. Only Sus activity in the embryo could explain, at least in part, the significant variability observed for both the RSS and the RRSS at each developmental stage. We conclude that Sus activity is a reliable marker of sink activity in developing pea seeds.     

Chloroplast structure and starch grain production as phylogenetic indicators in the lower Rhodophyceae

The position of starch grain production, the shape of the starch grains and the depth to which the pyrenoid is embedded in the chloroplast are used as indicators of evolution in the lower Rhodophyceae. A cell with cytoplasmic allantoid starch grains encasing the pyrenoid and no thylakoids between the chloroplast envelope and the pyrenoid is considered to be evolutionarily primitive. A cell with oval starch grains not associated with the pyrenoid and with a pyrenoid deeply embedded in the chloroplast is thought to be evolutionarily advanced. A polyphyletic origin of the Porphyridiales is discussed.     

Yeast fatty acid synthase: structure to function relationship

The yeast fatty acid synthase is a multifunctional enzyme composed of two nonidentical subunits in an alpha 6 beta 6 complex that is active in synthesizing fatty acids. The seven catalytic activities required for fatty acid synthesis are divided between the alpha and beta subunits such that the alpha 6 beta 6 complex has six complements of each activity. It has been proposed that these are organized into six centers for fatty acid synthesis. There are different opinions regarding the operation of these centers in the alpha 6 beta 6 complex, on view being that they are functionally independent and the other proposes half-sites activity for the complex. We have attempted to distinguish between these proposals by the most direct method of active site titration, i.e., quantitation of fatty acyl product in the absence of turnover. This was accomplished by using p-nitrophenyl thioacetate and thiophenyl malonate (in place of the coenzyme A analogues) as substrates along with NADPH, thereby depriving the yeast synthase of coenzyme A required to release product as fatty acyl coenzyme A. The amount of fatty acyl product formed was quantitated by gas-liquid chromatography, as well as by direct estimation of radioactivity in the product when p-nitrophenyl thio [1-14C] acetate was used as a substrate. In both cases, a stoichiometry of close to six was found for mole of fatty acid synthesized per mole of alpha 6 beta 6 complex. This indicates that there are six functional centers for fatty acid synthesis in the multifunctional yeast alpha 6 beta 6 fatty acid synthase and that these centers operate independently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of granule-bound starch synthase in determination of amylopectin structure and starch granule morphology in potato.

Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.     

Fungal catalases: Function, phylogenetic origin and structure

Most fungi have several monofunctional heme-catalases. Filamentous ascomycetes (Pezizomycotina) have two types of large-size subunit catalases (L1 and L2). L2-type are usually induced by different stressors and are extracellular enzymes; those from the L1-type are not inducible and accumulate in asexual spores. L2 catalases are important for growth and the start of cell differentiation, while L1 are required for spore germination. In addition, pezizomycetes have one to four small-size subunit catalases. Yeasts (Saccharomycotina) do not have large-subunit catalases and generally have one peroxisomal and one cytosolic small-subunit catalase. Small-subunit catalases are inhibited by substrate while large-subunit catalases are activated by H(2)O(2). Some small-subunit catalases bind NADPH preventing inhibition by substrate. We present a phylogenetic analysis revealing one or two events of horizontal gene transfers from Actinobacteria to a fungal ancestor before fungal diversification, as the origin of large-size subunit catalases. Other possible horizontal transfers of small- and large-subunit catalases genes were detected and one from bacteria to the fungus Malassezia globosa was analyzed in detail. All L2-type catalases analyzed presented a secretion signal peptide. Mucorales preserved only L2-type catalases, with one containing a secretion signal if two or more are present. Basidiomycetes have only L1-type catalases, all lacking signal peptide. Fungal small-size catalases are related to animal catalases and probably evolved from a common ancestor. However, there are several groups of small-size catalases. In particular, a conserved group of fungal sequences resemble plant catalases, whose phylogenetic origin was traced to a group of bacteria. This group probably has the heme orientation of plant catalases and could in principle bind NADPH. From almost a hundred small-subunit catalases only one fourth has a peroxisomal localization signal and in fact many fungi lack a peroxisomal catalase. Catalases have a deep buried active site and H(2)O(2) has to go through a long passage to reach it. In all known structures of catalases, the major channel has common features, particularly in the straight and narrow final section that is positioned perpendicular to the heme. Besides, other conserved channels are present in catalases whose function remains to be elucidated. One of these channels intercommunicates the major channels from the two R-related subunits. In three of the four known large-subunits catalase structures, the heme b is partially transformed into heme d. In Neurospora crassa, this occurs in vivo and is related to oxidative stress conditions in which singlet oxygen is produced. A pure source of singlet oxygen oxidizes catalases purified from different sources and singlet oxygen quenchers prevent oxidation. A second modification is observed in N. crassa catalase-1, in which the tyrosine that forms the fifth coordination bound to the heme iron makes a covalent bond with a vicinal cysteine, similarly to the tyrosine-histidine bonding found in Escherichia coli hydroperoxidase II. Molecular dynamics has been used to determine how H(2)O(2) reaches the enzyme active site and how products exit the protein. We found that the bottleneck of the major channel seems to disappear in water and is wide open in the presence of substrate. Amino acid residues exhibiting an increased residence time for H(2)O(2) are abundant at the protein surface and at the entrances to the major channel. The net effect of this is an increased H(2)O(2)/H(2)O ratio in the major channel. Once in the final section of this channel, H(2)O(2) is retained and tends to occupy specific sites while water molecules have a higher turnover rate and occupy different sites. Despite the intense study of catalases our knowledge of this enzyme is still limited and in need of new studies and different approaches.     

Granule-bound starch synthase (GBSSI) gene phylogeny of wild tomatoes (Solanum L. section Lycopersicon [Mill.] Wettst. subsection Lycopersicon)

Eight wild tomato species are native to western South America and one to the Galapagos Islands. Different classifications of tomatoes have been based on morphological or biological criteria. Our primary goal was to examine the phylogenetic relationships of all nine wild tomato species and closely related outgroups, with a concentration on the most widespread and variable tomato species Solanum peruvianum, using DNA sequences of the structural gene granule-bound starch synthase (GBSSI, or waxy). Results show some concordance with previous morphology-based classifications and new relationships. The ingroup comprised a basal polytomy composed of the self-incompatible green-fruited species S. chilense and the central to southern Peruvian populations of S. peruvianum, S. habrochaites, and S. pennellii. A derived clade contains the northern Peruvian populations of S. peruvianum (also self-incompatible, green-fruited), S. chmielewskii, and S. neorickii (self-compatible, green-fruited), and the self-compatible and red- to orange- to yellow-fruited species S. cheesmaniae, S. lycopersicum, and S. pimpinellifolium. Outgroup relationships are largely concordant with prior chloroplast DNA restriction site phylogenies, support S. juglandifolium and S. ochranthum as the closest outgroup to tomatoes with S. lycopersicoides and S. sitiens as basal to these, and support allogamy, self-incompatibility, and green fruits as primitive in the tomato clade.     

Polyketide synthase complexes: their structure and function in antibiotic biosynthesis.

This paper gives an overview of existing knowledge concerning the structure and deduced functions of polyketide synthases active in antibiotic-producing streptomycetes. Using monensin A as an example of a structurally complex polyketide metabolite, the problem of understanding how individual strains of microorganism are 'programmed' to produce a given polyketide metabolite is first outlined. The question then arises, how is the programming of polyketide assembly related to the structural organization of individual polyketide synthase complexes at the biochemical and genetic levels? Experimental results that help to illuminate these relations are described, in particular, those giving information about the structures and deduced functions of polyketide synthases involved in aromatic polyketide biosynthesis (actinorhodin, granaticin, tetracenomycin, whiE spore pigment and an act homologous region from the monensin-producing organism), as well as the macrolide polyketide synthase active in the biosynthesis of 6-deoxyerythronolide A.