Prediction and analysis of GH14 family ß-amylases in oat seedling extract: Structure and function insights using in silico approaches.

Oat (Avena sativa L.) seedling extract exhibited a high degree of catalytic activities. Bioinformatics were used to identify ß-amylases as abundant enzymes in the oat seedling extract. These identified oat enzymes are a member of the GH14 family. Proteins in the Avena sativa seedling extract were separated by SDS-PAGE and 2 major protein bands with an apparent molecular weights of 53 and 42 kDa were the subject of this study. These materials were digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS and database searches. These sequences were used to identify cDNAs from expressed sequence tags (EST) and Transcriptome Shotgun Assembly (TSA) of Avena sativa. Based upon EST and TSA sequences, at least 6 predicted different sequences were identified and assigned as ß-amylases. Insights into structural characterization of the oat predicted ß-amylases were analyzed using in silico approaches. The identified ß-amylases conserved the two Glu residues assigned as the "putative" catalytic residues, which would act as an acid and base pair in the catalytic process. A similar core (ß/α)8-barrel architecture was found in the predicted oat ß-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (ß/α)8-barrel domain. This suggests an accessibility of the non-reducing end of the substrate towards the oat ß-amylases and thus confirming that are exo-acting hydrolases. The results provide a detailed view of the main residues involved in catalysis in this kind of enzyme.

Evolution of the beta-amylase gene in the temperate grasses: Non-purifying selection, recombination, semiparalogy, homeology and phylogenetic signal.

Low-copy nuclear genes (LCNGs) have complex genetic architectures and evolutionary dynamics. However, unlike multicopy nuclear genes, LCNGs are rarely subject to gene conversion or concerted evolution, and they have higher mutation rates than organellar or nuclear ribosomal DNA markers, so they have great potential for improving the robustness of phylogenetic reconstructions at all taxonomic levels. In this study, our first objective is to evaluate the evolutionary dynamics of the LCNG ß-amylase by testing for potential pseudogenization, paralogy, homeology, recombination, and phylogenetic incongruence within a broad representation of the main Pooideae lineages. Our second objective is to determine whether ß-amylase shows sufficient phylogenetic signal to reconstruct the evolutionary history of the Pooid grasses. A multigenic (ITS, matK, ndhF, trnTL, and trnLF) tree of the study group provided a framework for assessing the ß-amylase phylogeny. Eight accessions showed complete absence of selection, suggesting putative pseudogenic copies or other relaxed selection pressures; resolution of Vulpia alopecuros 2x clones indicated its potential (semi) paralogy; and homeologous copies of allopolyploid species Festuca simensis, F. fenas, and F. arundinacea tracked their Mediterranean origin. Two recombination events were found within early-diverged Pooideae lineages, and five within the PACCMAD clade. The unexpected phylogenetic relationships of 37 grass species (26% of the sampled species) highlight the frequent occurrence of non-treelike evolutionary events, so this LCNG should be used with caution as a phylogenetic marker. However, once the pitfalls are identified and removed, the phylogenetic reconstruction of the grasses based on the ß-amylase exon+intron positions is optimal at all taxonomic levels.

Distribution and evolutionary dynamics of Stowaway Miniature Inverted repeat Transposable Elements (MITEs) in grasses.

The occurrence of Stowaway MITEs and their potential footprints in the grasses was assessed within an explicit phylogenetic framework. An organismal tree was used to analyze the distribution and evolutionary dynamics of these elements and their potential excision footprints in the fourth intron of the ß-amylase gene and in other introns of several nuclear genes across the Poaceae. Megablast and discontiguous megablast searches in the Entrez nucleotide database were performed for the ß-amylase, blz-1, dmc1, nuc, and xly genes MITEs. These elements and their potential footprints were distributed in introns and intergenic spacers of many other nuclear genes throughout the BEP lineages; however, they were absent in the studied PACCMAD lineages. A plausible underlying dynamic of successive acquisitions and deletions of ß-amylase Stowaway MITEs in the temperate grasses could be explained by three alternative hypotheses: (i) a single early acquisition of a palindrome element, similar to Tc1-Mariner, in the fourth intron of the ß-amylase gene in the ancestor of the Pooideae, followed by multiple independent losses, (ii) multiple independent acquisitions of MITEs in non-related pooid lineages or (iii) different waves of acquisition of MITEs, followed by multiple losses and horizontal transfers in the temperate grasses. This last hypothesis seems to fit best with the evidence found to date.

Characterization of a maize beta-amylase cDNA clone and its expression during seed germination.

A maize (Zea mays L.) cDNA clone (pZMB2) encoding beta-amylase was isolated from a cDNA library prepared from the aleurone RNA of germinating kernels. The cDNA encodes a predicted product of 488 amino acids with significant similarity to known beta-amylases from barley (Hordeum vulgare), rye (Secale cereale), and rice (Oryza sativa). Glycine-rich repeats found in the carboxyl terminus of the endosperm-specific beta-amylase of barley and rye are absent from the maize gene product. The N-terminal sequence of the first 20 amino acids of a beta-amylase peptide derived from purified protein is identical to the 5th through 24th amino acids of the predicted cDNA product, indicating the absence of a conventional signal peptide in the maize protein. Recombinant inbred mapping data indicate that the cDNA clone is single-copy gene that maps to chromosome 7L at position 83 centimorgans. Northern blot analysis and in vitro translation-immunoprecipitation data indicate that the maize beta-amylase is synthesized de novo in the aleurone cells but not in the scutellum during seed germination.