Identification of novel families of membrane proteins from the model plant Arabidopsis thaliana

MOTIVATION: The completion of the Arabidopsis genome offers the first opportunity to analyze all of the membrane protein sequences of a plant. The majority of integral membrane proteins including transporters, channels, and pumps contain hydrophobic alpha-helices and can be selected based on TransMembrane Spanning (TMS) domain prediction. By clustering the predicted membrane proteins based on sequence, it is possible to sort the membrane proteins into families of known function, based on experimental evidence or homology, or unknown function. This provides a way to identify target sequences for future functional analysis. RESULTS: An automated approach was used to select potential membrane protein sequences from the set of all predicted proteins and cluster the sequences into related families. The recently completed sequence of Arabidopsis thaliana, a model plant, was analyzed. Of the 25,470 predicted protein sequences 4589 (18%) were identified as containing two or more membrane spanning domains. The membrane protein sequences clustered into 628 distinct families containing 3208 sequences. Of these, 211 families (1764 sequences) either contained proteins of known function or showed homology to proteins of known function in other species. However, 417 families (1444 sequences) contained only sequences with no known function and no homology to proteins of known function. In addition, 1381 sequences did not cluster with any family and no function could be assigned to 1337 of these.     

Type III effector proteins from the plant pathogen Xanthomonas and their role in the interaction with the host plant

Pathogenicity of Xanthomonas campestris pathovar (pv.) vesicatoria and most other Gram-negative bacterial plant pathogens largely depends on a type III secretion (TTS) system which is encoded by hypersensitive response and pathogenicity (hrp) genes. These genes are induced in the plant and are essential for the bacterium to be virulent in susceptible hosts and for the induction of the hypersensitive response (HR) in resistant host and non-host plants. The TTS machinery secretes proteins into the extracellular milieu and effector proteins into the plant cell cytosol. In the plant, the effectors presumably interfere with cellular processes to the benefit of the pathogen or have an avirulence activity that betrays the bacterium to the plant surveillance system. Type III effectors were identified by their avirulence activity, co-regulation with the TTS system and homology to known effectors. A number of effector proteins are members of families, e.g., the AvrBs3 family in Xanthomonas. AvrBs3 localizes to the nucleus of the plant cell where it modulates plant gene expression. Another family that is also present in Xanthomonas is the YopJ/AvrRxv family. The latter proteins appear to act as SUMO cysteine proteases in the host. Here, we will present an overview about the regulation of the TTS system and its substrates and discuss the function of the AvrRxv and AvrBs3 family members in more detail.     

Isolation and characterization of extracellular α-galactosidases from Penicillium canescens

Two alpha-galactosidases were purified to homogeneity from the enzymatic complex of the mycelial fungus Penicillium canescens using chromatography on different sorbents. Substrate specificity, pH- and temperature optima of activity, stability under different pH and temperature conditions, and the influence of effectors on the catalytic properties of both enzymes were investigated. Genes aglA and aglC encoding alpha-galactosidases from P. canescens were isolated, and amino acid sequences of the proteins were predicted. In vitro feed testing (with soybean meal and soybean byproducts enriched with galactooligosaccharides as substrates) demonstrated that both alpha-galactosidases from P. canescens could be successfully used as feed additives. alpha-Galactosidase A belonging to the 27th glycosyl hydrolase family hydrolyzed galactopolysaccharides (galactomannans) and alpha-galactosidase C belonging to the 36th glycosyl hydrolase family hydrolyzed galactooligosaccharides (stachyose, raffinose, etc.) of soybean with good efficiency, thus improving the digestibility of fodder.     

Molecular cloning and expression of a novel family A endoglucanase gene from Fibrobacter succinogenes S85 in Escherichia coli

A Fibrobacter succinogenes S85 gene that encodes endoglucanase hydrolysing CMC and xylan was cloned and expressed in Escherichia coli DH5 by using pUC19 vector. Recombinant plasmid DNA from a positive clone hydrolysing CMC and xylan was designated as pCMX1, harboring 2,043 bp insert. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The nucleotide sequence accession number of the cloned gene sequence in Genbank is U94826. The endoglucanase gene cloned in this study does not have amino sequence homology to the other endoglucanase genes from F. succinogenes S85, but does show sequence homology to family 5 (family A) of glycosyl hydrolases from several species. The ORF encodes a polypeptide of 654 amino acids with a measured molecular weight of 81.3 kDa on SDS-PAGE. Putative signal sequences, Shine-Dalgarno-type ribosomal binding site and promoter sequences (-10) related to the consensus promoter sequences were deduced. The recombinant endoglucanase by E. coli harboring pCMX1 was partially purified and characterized. N-terminal sequences of endoglucanase were Ala-Gln-Pro-Ala-Ala, matched with deduced amino sequences. The temperature range and pH for optimal activity of the purified enzyme were 55 approximately 65 degrees C and 5.5, respectively. The enzyme was most stable at pH 6 but unstable under pH 4 with a K(m) value of 0.49% CMC and a V(max) value of 152 U/mg.     

Cloning and expression of a thermostable alpha-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris

The first gene (alpha-gal1) encoding an extracellular alpha-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The alpha-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal alpha-galactosidases belonging to glycosyl hydrolase family 27. The alpha-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant alpha-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at 70 degrees C, pH 4.5, and lost no activity over 10 days at 50 degrees C. alpha-Gal1 followed Michaelis-Menten kinetics (Vmax of 240.3 micronM/min/mg, Km of 0.294 mM) and was inhibited competitively by galactose (Km obs of 0.57 mM, Ki of 2.77 mM). The recombinant T. emersonii alpha-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the alpha-galactosidic linkage. Owing to its substrate preference and noteworthy stability, alpha-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.     

Defense peptides of plant immune system

Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms to combat pathogens. They are important effector molecules of the immune system both in animals and plants. AMPs are diverse in structure and mode of action. Based on homology of amino acid sequences and 3D structures several AMP families have been distinguished. They are defensins, thionins, lipid transfer proteins, hevein- and knottin-like peptides, and cyclotides. AMPs display broad-spectrum antimicrobial activity and thus show promise for the development of disease- resistant crops by genetic engineering and for the production of new-generation drugs. In this paper, the properties of the main AMP families (defensins and hevein-like peptides) and of a new 4-Cys plant AMP family are reviewed.     

Defense peptides of plant immunity

Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms to fight pathogens. They are important effector molecules of the immune system both in animals and plants. AMPs are diverse in structure and mode of action. Based on the homology of amino acid sequences and 3D structures several AMP families have been distinguished. They are defensins, thionins, lipid transfer proteins, hevein- and knottin-like peptides, and cyclotides. AMPs display broad-spectrum antimicrobial activity and thus show promise for the development of disease-resistant crops by genetic engineering and for the production of new-generation drugs. In this paper, the properties of the main AMP families (defensins and hevein-like peptides) and of new 4-Cys plant AMP family are reviewed.     

Evolution of mammalian chitinase(-like) members of family 18 glycosyl hydrolases

Family 18 of glycosyl hydrolases encompasses chitinases and so-called chi-lectins lacking enzymatic activity due to amino acid substitutions in their active site. Both types of proteins widely occur in mammals although these organisms lack endogenous chitin. Their physiological function(s) as well as evolutionary relationships are still largely enigmatic. An overview of all family members is presented and their relationships are described. Molecular phylogenetic analyses suggest that both active chitinases (chitotriosidase and AMCase) result from an early gene duplication event. Further duplication events, followed by mutations leading to loss of chitinase activity, allowed evolution of the chi-lectins. The homologous genes encoding chitinase(-like) proteins are clustered in two distinct loci that display a high degree of synteny among mammals. Despite the shared chromosomal location and high homology, individual genes have evolved independently. Orthologs are more closely related than paralogues, and calculated substitution rate ratios indicate that protein-coding sequences underwent purifying selection. Substantial gene specialization has occurred in time, allowing for tissue-specific expression of pH optimized chitinases and chi-lectins. Finally, several family 18 chitinase-like proteins are present only in certain lineages of mammals, exemplifying recent evolutionary events in the chitinase protein family.     

Cloning and expression of the gene encoding <Emphasis Type="BoldItalic">Streptomyces coelicolor</Emphasis> A3(2) α-galactosidase belonging to family 36

The α-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus thermophilus was over 40%. The α-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 °C and pH 7. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates.     

Microbial carbohydrate esterases deacetylating plant polysaccharides

Several plant polysaccharides are partially esterified with acetic acid. One of the roles of this modification is protection of plant cell walls against invading microorganisms. Acetylation of glycosyl residues of polysaccharides prevents hydrolysis of their glycosidic linkages by the corresponding glycoside hydrolases. In this way the acetylation also represents an obstacle of enzymatic saccharification of plant hemicelluloses to fermentable sugars which appears to be a hot topic of current research. We can eliminate this obstacle by alkaline extraction or pretreatment leading to saponification of ester linkages. However, this task has been accomplished in a different way in the nature. The acetyl groups became targets of microbial carbohydrate esterases that evolved to overcome the complexity of the plant cell walls and that cooperate with glycoside hydrolases in plant polysaccharide degradation. This article concentrates on enzymes deacetylating plant hemicelluloses excluding pectin. They are currently grouped in at least 8 families, specifically in CE families 1–7 and 16, originally assigned as acetylxylan esterases, the enzymes acting on hardwood acetyl glucuronoxylan and its fragments generated by endo-β-1,4-xylanases. There are esterases deacetylating softwood galactoglucomannan, but they have not been classified yet. The enzymes present in CE families 1–7 differ in structure and substrate and positional specificity. There are families behaving as endo-type and exo-type deacetylates, i.e. esterases deacetylating internal sugar residues of partially acetylated polysaccharides and also esterases deacetylating non-reducing end sugar residues in oligosaccharides. With one exception, the enzymes of all mentioned CE families belong to serine type esterases. CE family 4 harbors enzymes that are metal-dependent aspartic esterases. Three-dimensional structures have been solved for members of the first seven CE families, however, there is still insufficient knowledge about their substrate specificity and real physiological role. Current knowledge on catalytic properties of the selected families of CEs is summarized in this review. Some of the families are emerging also as new biocatalysts for regioselective acylation and deacylation of carbohydrates.     

DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity

Acyl CoA:diacylgycerol acyltransferase (EC; DGAT) catalyzes the final step in the production of triacylglycerol. Two polypeptides, which co-purified with DGAT activity, were isolated from the lipid bodies of the oleaginous fungus Mortierella ramanniana with a procedure consisting of dye affinity, hydroxyapatite affinity, and heparin chromatography. The two enzymes had molecular masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and showed a broad activity maximum between pH 6 and 8. Based on partial peptide sequence information, polymerase chain reaction techniques were used to obtain full-length cDNA sequences encoding the purified proteins. Expression of the cDNAs in insect cells conferred high levels of DGAT activity on the membranes isolated from these cells. The two proteins share 54% homology with each other but are unrelated to the previously identified DGAT gene family (designated DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene family, or to any other gene family with ascribed function. This report identifies a new gene family, including members in fungi, plants and animals, which encode enzymes with DGAT function. To distinguish the two unrelated families we designate this new class DGAT2 and refer to the M. ramanniana genes as MrDGAT2A and MrDGAT2B.     

Recommendations for naming plant pathogenesis-related proteins

Pathogenesis-related proteins (abbreviated PRs) are defined as plant proteins that are induced in pathological or related situations. We propose a unifying nomenclature for PRs based on their grouping into families sharing amino acid sequences, serological relationship, and/or enzymatic or biological activity. The nomenclature classifies novel proteins identified by electrophoresis or chromatography along with those established by other workers. The previously proposed system of the five well-established families from tobacco is extended to accommodate a further six families. Families are indicated by arabic numerals and individual members are named by lower case letters in the order in which they are described. Additional rules are proposed to deal with forms containing more than a single polypeptide and as yet unclassified PRs. PR genes whose sequences are conserved but whose designations are not based on function are to be designated Ypr in accordance with the recommendations of the Commission on Plant Gene Nomenclature.     

A comparative analysis of two cDNA clones of the cellulase gene family from anaerobic fungus Piromyces rhizinflata

Cellulase family and some other glycosyl hydrolases of anaerobic fungi inhabiting the digestive tract of ruminants are believed to form an enzyme complex called cellulosome. Study of the individual component of cellulosome may shed light on understanding the organization of this complex and its functional mechanism. We have analysed the primary sequences of two cellulase clones, cel5B and cel6A, isolated from the cDNA library of ruminal fungus, Piromyces rhizinflata strain 2301. The deduced amino acid sequences of the catalytic domain of Cel5B, encoded by cel5B, showed homology with the subfamily 4 of the family 5 (subfamily 5(4)) of glycosyl hydrolases, while cel6A encoded Cel6A belonged to family 6 of glycosyl hydrolases. Phylogenetic tree analysis suggested that the genes of subfamily 5(4) glycosyl hydrolases of P. rhizinflata might have been acquired from rumen bacteria. Cel5B and Cel6A were modular enzymes consisting of a catalytic domain and dockerin domain(s), but not a cellulose binding domain. The occurrence of dockerin domains indicated that both enzymes were cellulosome components. The catalytic domain of the Cel5B (Cel5B') and Cel6A (Cel6A') recombinant proteins were purified. The optimal activity conditions with carboxymethyl cellulose (CMC) as the substrate were pH 6.0 and 50 degrees C for Cel5B', and pH 6.0 and 37-45 degrees C for Cel6A'. Both Cel5B' and Cel6A' exhibited activity against CMC, barley beta-glucan, Lichenan, and oat spelt xylan. Cel5B' could also hydrolyse p-nitrophenyl-beta-d-cellobioside, Avicel and filter paper while Cel6A' did not show any activity on these substrates. It is apparent that Cel6A' acted as an endoglucanase and Cel5B' possessed both endoglucanase and exoglucanase activities. No synergic effect was observed for these recombinant enzymes in vitro on Avicel and CMC.     

Transgenic expression of plant chitinases to enhance disease resistance

Crop plants have evolved an array of mechanisms to counter biotic and abiotic stresses. Many pathogenesis-related proteins are expressed by plants during the attack of pathogens. Advances in recombinant DNA technology and understanding of plant–microbe interactions at the molecular level have paved the way for isolation and characterization of genes encoding such proteins, including chitinases. Chitinases are included in families 18 and 19 of glycosyl hydrolases (according to www.cazy.org) and they are further categorized into seven major classes based on their aminoacid sequence homology, three-dimensional structures, and hydrolytic mechanisms of catalytic reactions. Although chitin is not a component of plant cell walls, plant chitinases are involved in development and non-specific stress responses. Also, chitinase genes sourced from plants have been successfully over-expressed in crop plants to combat fungal pathogens. Crops such as tomato, potato, maize, groundnut, mustard, finger millet, cotton, lychee, banana, grape, wheat and rice have been successfully engineered for fungal resistance either with chitinase alone or in combination with other PR proteins.     

High Phylogenetic Diversity of Glycosyl Hydrolase Family 10 and 11 Xylanases in the Sediment of Lake Dabusu in China

Soda lakes are one of the most stable naturally occurring alkaline and saline environments, which harbor abundant microorganisms with diverse functions. In this study, culture-independent molecular methods were used to explore the genetic diversity of glycoside hydrolase (GH) family 10 and GH11 xylanases in Lake Dabusu, a soda lake with a pH value of 10.2 and salinity of 10.1%. A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences. Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi. A full-length GH10 xylanase gene (xynAS10-66) was directly cloned and expressed in Escherichia coli, and the recombinant enzymes showed high activity at alkaline pH. These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.