The present study was conducted to determine the gene responsible for beta-glucosidase (BGL) production and to generate a full-length complementary DNA (cDNA) of one of the putative BGL genes, which showed a significant expression level when Schizophyllum commune KUC9397 was grown in optimized medium. The relative expression levels of seven genes encoding BGL of S. commune KUC9397 were determined with real-time quantitative reverse transcription PCR in cellulose-containing optimized medium (OM) compared to glucose-containing basal medium (BM). The most abundant transcript was bgl3a in OM. The transcript number of the bgl3a increased more than 57.60-fold when S. commune KUC9397 was grown on cellulose-containing OM compared to that on glucose-containing BM. The bgl3a was identified, and a deduced amino acid sequence of bgl3a shared homology (97%) with GH3 BGL of S. commune H4-8. This is the first report showing the transcription levels of genes encoding BGL and identification of full-length cDNA of glycoside hydrolase 3 (GH3) BGL from S. commune. Furthermore, this study is one of the steps for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

• MeSH
• aktivace transkripce MeSH
• beta-glukosidasa biosyntéza   genetika   MeSH
• kultivační média chemie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• molekulární sekvence - údaje MeSH
• Schizophyllum enzymologie   genetika   růst a vývoj   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• stanovení celkové genové exprese * MeSH
• upregulace MeSH
• Publikační typ
• časopisecké články MeSH

Bacteria represent an underexplored source of diglycosidases. Twenty-five bacterial strains from the genera Actinoplanes, Bacillus, Corynebacterium, Microbacterium, and Streptomyces were selected for their ability to grow in diglycosylated flavonoids-based media. The strains Actinoplanes missouriensis and Actinoplanes liguriae exhibited hesperidin deglycosylation activity (6-O-α-L-rhamnosyl-β-D-glucosidase activity, EC 3.2.1.168), which was 3 to 4 orders of magnitude higher than the corresponding monoglycosidase activities. The diglycosidase production was confirmed in A. missouriensis by zymographic assays and NMR analysis of the released disaccharide, rutinose. The gene encoding the 6-O-α-L-rhamnosyl-β-D-glucosidase was identified in the genome sequence of A. missouriensis 431(T) (GenBank accession number BAL86042.1) and functionally expressed in Escherichia coli. The recombinant protein hydrolyzed hesperidin and hesperidin methylchalcone, but not rutin, which indicates its specificity for 7-O-rutinosylated flavonoids. The protein was classified into the glycoside hydrolase family 55 (GH55) in contrast to the known eukaryotic diglycosidases, which belong to GH1 and GH5. These findings demonstrate that organisms other than plants and filamentous fungi can contribute to an expansion of the diglycosidase toolbox.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• beta-glukosidasa genetika   metabolismus   MeSH
• chalkonoidy chemie   metabolismus   MeSH
• disacharidy chemie   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• flavonoidy chemie   metabolismus   MeSH
• fylogeneze MeSH
• glykosidy chemie   metabolismus   MeSH
• hesperidin analogy a deriváty   chemie   metabolismus   MeSH
• hydrolýza MeSH
• klonování DNA MeSH
• Micromonosporaceae klasifikace   genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• rhamnosa chemie   metabolismus   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

• MeSH
• aktivace enzymů MeSH
• beta-glukosidasa genetika   metabolismus   MeSH
• databáze proteinů MeSH
• genová knihovna MeSH
• hydrolýza MeSH
• kodon MeSH
• kukuřice setá genetika   metabolismus   MeSH
• mutageneze MeSH
• proteinové inženýrství * metody   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• substrátová specifita MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The maize β-D-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-β-D-glucopyranoside versus the trans-zeatin-O-β-D-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-β-D-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.

• MeSH
• aminokyseliny metabolismus   MeSH
• beta-glukosidasa chemie   genetika   MeSH
• cytokininy metabolismus   MeSH
• glukosidy metabolismus   MeSH
• hydrolýza MeSH
• isomerie MeSH
• kukuřice setá chemie   enzymologie   genetika   MeSH
• molekulární konformace MeSH
• mutace MeSH
• mutageneze cílená metody   MeSH
• rostlinné geny MeSH
• rostlinné proteiny chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zeatin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize β-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.

• MeSH
• beta-glukosidasa genetika   metabolismus   MeSH
• chloroplasty účinky léků   metabolismus   MeSH
• cytokininy metabolismus   MeSH
• fazol genetika   MeSH
• geneticky modifikované rostliny MeSH
• glukosidy metabolismus   MeSH
• hybridizace genetická MeSH
• kukuřice setá enzymologie   genetika   MeSH
• semenáček cytologie   účinky léků   růst a vývoj   MeSH
• tabák cytologie   účinky léků   genetika   metabolismus   MeSH
• vakuoly účinky léků   genetika   metabolismus   MeSH
• zeatin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Moonlighting proteins have two different functions within a single polypeptide chain. Exploring moonlighting enzymes from the environment using the metagenomic approach is interesting. In the present study, a novel β-glucosidase gene, designated as bgl1D, with lipolytic activity (renamed Lip1C) was cloned through function-based screening of a metagenomic library from uncultured soil microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis also indicated that Lip1C and other putative lipases are closely related. Biochemical characterization demonstrated that the maximum activity of the recombinant Lip1C protein occurs at pH 8.0 and 30°C using 4-nitrophenyl butyrate as substrate. The putative lipase had an apparent K(m) value of 0.88 mmol/L, a k(cat) value of 212/min, and a k(cat)/K(m) value of 241 L/mmol/min. Lip1C exhibited habitat-specific characteristics with 5 mmol/L AlCl(3), CuCl(2), and LiCl. The characterization of the biochemical properties of Lip1C enhances our understanding of this novel moonlighting enzyme isolated from a soil metagenome.

• MeSH
• Bacteria klasifikace   enzymologie   genetika   izolace a purifikace   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• beta-glukosidasa chemie   genetika   metabolismus   MeSH
• fylogeneze MeSH
• kinetika MeSH
• lipasa chemie   genetika   metabolismus   MeSH
• metagenom MeSH
• molekulární sekvence - údaje MeSH
• půdní mikrobiologie MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• beta-glukosidasa genetika   chemie   metabolismus   MeSH
• cystein genetika   MeSH
• kukuřice setá enzymologie   MeSH
• mutageneze cílená MeSH
• vztahy mezi strukturou a aktivitou genetika   MeSH

Zm-p60.1 is maize cDNA coding cytokinin-glucoside specific beta-glucosidase. Indigogenic method was used for histochemical localization of Zm-p60.1 beta-glucosidase activity in various developmental stages of transgenic tobacco anthers. Expression of Zm-p60.1 cDNA in T7 tobacco plants is controlled by the CaMV 35S promoter. Another type of tobacco transformant expresses Zm-p60.1 under the control of LAT 52 promoter. Histochemical detection has proved different patterns of beta-glucosidase activity during tobacco pollen development in these two types of transformants. Zm-p60.1 beta-glucosidase activity had not direct influence on pollen germinability.

• MeSH
• beta-glukosidasa genetika   MeSH
• DNA primery genetika   MeSH
• exprese genu MeSH
• geneticky modifikované rostliny MeSH
• jedovaté rostliny MeSH
• kukuřice setá enzymologie   genetika   růst a vývoj   MeSH
• messenger RNA genetika   metabolismus   MeSH
• pyl enzymologie   genetika   růst a vývoj   MeSH
• RNA rostlin genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• tabák MeSH
• transformace genetická MeSH