Demonstration of Activity of α‐Galactosidase Secreted by Cucumis sativus L. Cells

A simple, rapid and reproducible procedure for the identification of extracellular cucumber (Cucumis sativus L.) α‐galactosidase is described using callus cultures of seedlings from the tested plant, hairy roots of 2‐day‐old seedlings of cucumber germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of the intracellular and extracellular activities of α‐galactosidase, 6‐bromo‐2‐naphthyl‐α‐D‐galactopyranoside and p‐nitrophenyl‐α‐D‐galactopyranoside, respectively, were used as synthetic substrates. The extracellular α‐galactosidase activity was identified by evaluating the dye‐zones in agar medium. The enzyme from cucumber callus cultures and seedling roots, cultivated on agar plates supplemented with 6‐bromo‐2‐naphthyl‐α‐D‐galactopyranoside, hydrolyzed this substrate releasing 6‐bromo‐2‐naphthol. By simultaneous coupling with hexazonium p‐rosaniline the corresponding azodye was formed. Thus, the extracellular enzyme was detected by the presence of reddish‐brown zones on the agar plates around the plant material. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 44.6% intracellular and 55.4% extracellular distribution of α‐galactosidase activity. The described agar plate method enables a rapid, simple and specific detection of plant producers of extracellular α‐galactosidase.     

An inventory of peptide hydrolases and arylamidases in Flavobacterium II b

The determination of the number and the specificity of peptide hydrolases and arylamidases present in a cell-free sonicate from Flavobacterium II b, a species phenotypically similar to Flavobacterium balustinum , involved polyacrylamide gel electrophoresis and coloration of end products or staining procedures using diazotation reactions. Only substrates with L-configuration amino acids and dipeptides with an unsubstituted R_N-group were cleaved. One protein band, that was able to hydrolyse skimmed milk agar, was observed but carboxypeptidase and endopep-tidase (chymotrypsin and trypsin activities) were absent. Zymograms exhibited ary-lamidase, dipeptidase and tripeptidase activities. Results underline the distinction between the L-leucine aminopeptidase and the L-leucylarylamidase.     

A novel Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria has β‐hydroxylating activity of N‐succinyl l‐leucine

An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

Growth and biochemical characterization of associations between cyanobionts and wheat seedlings in co-culturing experiments

N₂-fixing cyanobacteria are unique in their capacity to form symbiotic associations with a wide range of eukaryotic hosts belonging to different plant groups. The present study was undertaken to analyze the interactions of the cyanobiont PI 01 (from Azolla pinnata) and Nostoc PCC 9229 (from Gunnera monoika) with wheat seedlings, in co-culturing experiments. Each of the cyanobionts enhanced significantly the volume of root and shoot biomass in the experimental cultures. The transverse sections of roots in the co-cultured seedlings revealed the presence of aseriate packets of cyanobionts below the root epidermis. The investigated cyanobionts excreted amino acids (His, Met, Val) and sugars into the medium, while indoleacetic acid was detected when the cyanobionts were grown in a tryptophan containing medium. During the co-culturing, sugars and proline were detected in the extracellular filtrates. It can be hypothesized that these sugars and amino acids may serve as signal substances in the development of functional associations between the relevant cyanobionts and the wheat seedlings.     

Involvement of exopeptidases in dehydration tolerance of spring wheat seedlings

The observed inability of 6-d-old seedlings of spring wheat (Triticum aestivum L.) to tolerate the same water deficit as compared to the 4-d-old seedlings seems to be associated with the higher carboxypeptidase and lower aminopeptidase activities. Free amino acid pools differentiated also the 4-d-old seedlings from the older ones. Dehydration decreased the amino acid content in 4-d-old seedlings, increased it in 6-d-old seedlings and changed composition of amino acid pool. In tolerant phase of wheat seedling growth carboxypeptidase activity increased in response to water deficit and aminopeptidase activity increased in dehydrated seedlings, independently of their age.     

Incorporation of β‐amino acids into ascidiacyclamides: Effects on conformation,cytotoxicity and interaction with copper (II) ion

Seven ascidiacyclamide [cyclo(–Ile–oxazoline–d ‐Val–thiazole–)₂] (ASC) analogues incorporating the β‐amino acids βIle, βoxazoline, and/or d ‐βVal were synthesized. We then investigated the effects of the position and number of incorporated β‐amino acids on the structure, cytotoxicity, and copper binding by these seven analogues. The structural analyses revealed that both βIle and d ‐βVal favor a gauche‐type θ torsion angles, while βoxazoline favors a trans‐type θ torsion angle. Expansion of the macrocycle by incorporation of βIle or d ‐βVal readily induced molecular folding. On the other hand, the incorporation of two βoxazoline residues strongly extended the peptide conformation, and the incorporation of one was sufficient for the moderate restriction important for conformational equilibrium and cytotoxicity. Despite expansion of the macrocycles, the structure‐cytotoxicity relationships were largely maintained. In studies of complexation of the analogues with Cu (II) ion, the position and number of incorporated β‐amino acids had a large impact on the structure of the metal complex and may contribute to its stabilization.     

A structural motif is the recognition site for a new family of bacterial protein O-glycosyltransferases

The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA‐I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDA‐associated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA‐I, which comprises imperfect repeats of a 19‐amino‐acid consensus sequence and is predicted to form a β‐helix. Here, we show that Aah homologues can be found in many Gram‐negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA‐like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19‐amino‐acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of β‐helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a β‐helical polypeptide lacking the 19‐amino‐acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O‐glycosyltransferases that modify various substrates recognized through a structural motif.     

Comparison of aminopeptidase inhibition by amino acids in human and porcine skeletal muscle tissues in vitro

We compared the inhibitory action of individual amino acids in vitro on the activities of alanyl-, arginyl-, leucyl- and pyroglutamyl aminopeptidases purified from human and porcine skeletal muscle tissues. The range of susceptibility to inhibition by individual amino acids (<25 mM) for different aminopeptidase types broadly paralleled that for the respective substrate specificities (in terms of relative rates of hydrolysis of amino acyl-AMC derivatives) for these enzymes. Thus, alanyl aminopeptidase (which hydrolyses a broad range of aminoacyl-AMC substrates) was inhibited by a correspondingly broad range of amino acids (although the respective ranking order of amino acids was not identical in each case), whereas pyroglutamyl aminopeptidase (which hydrolyses only pyroglutamyl AMC as substrate) was inhibited by pyroglutamic acid only. The mode of inhibition (competitive/non-competitive) varied for different enzyme types, both within and between each species. For enzymes purified from human muscle, alanyl, arginyl and leucyl aminopeptidases were inhibited by amino acids via the non-competitive mode (pyroglutamyl aminopeptidase via the competitive mode), whereas corresponding enzymes purified from porcine muscle were inhibited via the competitive mode. The data obtained indicate that the same aminopeptidase types are present in human and porcine skeletal muscle tissues, with corresponding enzymes having broadly similar assay characteristics and susceptibilities to inhibition by amino acids (although the mode of inhibition for corresponding enzymes may differ in each species). Such data obtained in vitro may prove of value in devising experimental strategies to manipulate protein turnover/muscle deposition in vivo, via inhibition of aminopeptidase action after administration of an appropriate admixture of amino acids.     

An amino acid code for β‐sheet packing structure

To understand the relationship between protein sequence and structure, this work extends the knob‐socket model in an investigation of β‐sheet packing. Over a comprehensive set of β‐sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the two types of four‐residue packing cliques necessary to describe β‐sheet packing were characterized. Both occur between two adjacent hydrogen bonded β‐strands. First, defining the secondary structure packing within β‐sheets, the combined socket or XY:HG pocket consists of four residues i, i+2 on one strand and j, j+2 on the other. Second, characterizing the tertiary packing between β‐sheets, the knob‐socket XY:H+B consists of a three‐residue XY:H socket (i, i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, two types of knob‐sockets are found: side‐chain and main‐chain sockets. The amino acid composition of the pockets and knob‐sockets reveal the sequence specificity of β‐sheet packing. For β‐sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side‐chain and main‐chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β‐sheet structure and provide an intuitive topological mapping of β‐sheet packing. Proteins 2014; 82:2128–2140. © 2014 Wiley Periodicals, Inc.     

Polyglycine hydrolases: Fungal β‐lactamase‐like endoproteases that cleave polyglycine regions within plant class IV chitinases

Polyglycine hydrolases are secreted fungal proteases that cleave glycine–glycine peptide bonds in the inter‐domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es‐cmp) and Cochliobolus carbonum (Bz‐cmp). Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. Peptides from a tryptic digest of Es‐cmp were analyzed by LC‐MS/MS and the spectra obtained were matched to a draft genome sequence of E. sorghi. From this analysis, a 642 amino acid protein containing a predicted β‐lactamase catalytic region of 280 amino acids was identified. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. carbonum from their cDNAs. Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS‐PAGE and MALDI‐MS based assays. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es‐cmp to glycine, resulting in loss of catalytic activity. BLAST searching of publicly available fungal genomes identified full‐length homologous proteins in 11 other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the β‐lactamase‐like region. This is the first report of a predicted β‐lactamase that is an endoprotease.     

Crystal structure of the secreted protein HP1454 from the human pathogen Helicobacter pylori

HP1454 is a protein of 303 amino acids found in the extracellular milieu of Helicobacter pylori. The protein structure, crystallized in the orthorhombic C222₁ space group with one molecule per asymmetric unit, has been determined using the single‐wavelength anomalous dispersion method. HP1454 exhibits an elongated bent shape, composed of three distinct domains. Each domain possesses a fold already present in other structures: Domain I contains a three‐strand antiparallel β‐barrel flanked by a long α‐helix, Domain II is an anti‐parallel three‐helix bundle, and Domain III a β‐sheet flanked by two α‐helices. The overall assembly of the protein does not bear any similarity with known structures. Proteins 2014; 82:2868–2873. © 2014 Wiley Periodicals, Inc.     

EXTRACELLULAR ENZYMES OF THE MICROCYSTIS AERUGINOSA PCC 7813 STRAIN ARE INHIBITED IN THE PRESENCE OF HYDROQUINONE AND PYROGALLOL,ALLELOCHEMICALS PRODUCED BY AQUATIC PLANTS1

Several cyanobacterial species have a high potential to dominate in marine environments and freshwater reservoirs, and the ecological and physiological reasons for this phenomenon are not understood comprehensively. In this study, the ability of a Microcystis aeruginosa Kütz. strain to produce free dissolved enzymes was documented. We have observed that this highly toxic strain releases alkaline phosphatase, leucine aminopeptidase, and β‐glucosidase into the ambient environment. Additionally, the inhibitory activity of selected phenols produced by aquatic plants on the activity of these enzymes was analyzed. The investigated compounds, pyrogallol and, to a lesser degree, hydroquinone, decreased the activity of extracellular enzymes produced by M. aeruginosa, with leucine aminopeptidase being the most sensitive to the inhibitors. The noncompetitive character of enzymatic inhibition suggests that the polyphenols produced by aquatic plants are able to influence the activity of different extracellular or membrane‐bound enzymes.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Moisture content and nutrition as selection forces for emerald ash borer larval feeding behaviour

1. The exotic phloem‐feeding emerald ash borer (EAB), Agrilus planipennis, has killed tens of millions of North American ash trees (Fraxinus) since its first detection in the U.S.A. in 2002. Ash trees are killed by larval feeding in the cambial region, which disrupts translocation of photosynthates and nutrients. 2. We observed that EAB larvae feed predominantly downwards in naturally grown green ash trees, a behaviour confirmed in greenhouse‐grown black ash seedlings. Furthermore, biomass of larvae feeding downwards was greater than that for larvae feeding upwards. 3. We sought to determine the relative importance of four selection forces (i.e. gravity, moisture content, plant defence, and nutrition) in driving this downward feeding behaviour in this study. The gravity and plant defence (i.e. polyphenols) hypotheses were ruled out because even when seedlings were grown upside down, more EAB larvae moved upwards (towards the root area), and phloem tissue below the feeding site contained higher concentrations of defensive compounds than that above the feeding site. 4. The moisture content hypothesis was supported as phloem moisture above the feeding site decreased to levels reducing survivorship and biomass but was unaffected below. The nutrition hypothesis was also supported as the levels of 11 amino acids (mostly essential amino acids) below the feeding site were greater than those above. Furthermore, growth of larvae reared on an artificial diet deficient in protein and amino acids was worse than larvae reared in diet with complete ingredient or diet deficient in either protein or amino acids. 5. We conclude that moisture content and nutrients are two selective forces for the downward feeding behaviour of EAB larvae.     

Seasonal changes in the free amino acids of oat and barley phloem sap in relation to plant growth stage and growth of Rhopalosiphum padi

The concentrations and composition of free amino acids in phloem sap from two cultivars of oats and barley, both susceptible to the aphid Rhopalosiphum padi, were determined by means of high performance liquid chromatography. Sap was collected from excised aphid stylets at three developmental stages (seedlings, tillering plants and plants undergoing stem elongation) from plants given or not given fertiliser and grown outdoors. In connection, the growth of individual R. padi nymphs was estimated at the same phenological stages on plants grown in the greenhouse. The content of free amino acids was consistently higher in seedlings than in plants at the early tillering stage. Only in seedlings did the addition of fertiliser increase amino acid levels. Barley phloem sap contained more free amino acids than that of oats when fertiliser was added and at later developmental stages. Phloem sap of oats and barley showed similar patterns in their composition of free amino acids at the seedling stage, but as the plants grew older the patterns became increasingly different. Plants given fertiliser had higher amounts of dicarboxylic amino acids (glutamic and aspartic acid) than unfertilised plants. The concentrations of γ-amino butyric acid, glycine, histidine, and methionine were very low in all treatments. The relative growth rates of R. padi nymphs were low when amino acid content was low and vice versa. The results are discussed in relation to host plant suitability and plant resistance mechanisms.     

A peroxisomal thioesterase plays auxiliary roles in plant β‐oxidative benzoic acid metabolism

Peroxisomal β‐oxidative degradation of compounds is a common metabolic process in eukaryotes. Reported benzoyl‐coenzyme A (BA‐CoA) thioesterase activity in peroxisomes from petunia flowers suggests that, like mammals and fungi, plants contain auxiliary enzymes mediating β‐oxidation. Here we report the identification of Petunia hybrida thioesterase 1 (PhTE1), which catalyzes the hydrolysis of aromatic acyl‐CoAs to their corresponding acids in peroxisomes. PhTE1 expression is spatially, developmentally and temporally regulated and exhibits a similar pattern to known benzenoid metabolic genes. PhTE1 activity is inhibited by free coenzyme A (CoA), indicating that PhTE1 is regulated by the peroxisomal CoA pool. PhTE1 downregulation in petunia flowers led to accumulation of BA‐CoA with increased production of benzylbenzoate and phenylethylbenzoate, two compounds which rely on the presence of BA‐CoA precursor in the cytoplasm, suggesting that acyl‐CoAs can be exported from peroxisomes. Furthermore, PhTE1 downregulation resulted in increased pools of cytoplasmic phenylpropanoid pathway intermediates, volatile phenylpropenes, lignin and anthocyanins. These results indicate that PhTE1 influences (i) intraperoxisomal acyl‐CoA/CoA levels needed to carry out β‐oxidation, (ii) efflux of β‐oxidative products, acyl‐CoAs and free acids, from peroxisomes, and (iii) flux distribution within the benzenoid/phenylpropanoid metabolic network. Thus, this demonstrates that plant thioesterases play multiple auxiliary roles in peroxisomal β‐oxidative metabolism.     

Induced phenylamide accumulation in response to pathogen infection and hormone treatment in rice (Oryza sativa)

Rice plants accumulate various specialized metabolites, including phenylamides, in response to pathogen attack. We prepared 25 phenylamides, and developed a method of analyzing them by multiple reaction monitoring with liquid chromatography coupled with tandem mass spectrometry. We analyzed phenylamides in rice leaves infected with Cochliobolus miyabeanus and Xanthomonas oryzae. The phenylamides induced included benzoyltryptamine, cinnamoyl-, p-coumaroyl-, feruloyl-, and benzoylserotonins, cinnamoyl and benzoyltyramines, feruloylagmatine, and feruloylputrescine. Some of the phenylamides exhibited antimicrobial activity against C. miyabeanus and X. oryzae, indicating that they are phytoalexins. Treatment with jasmonic acid, salicylic acid, 6-benzylaminopurine, and ethephone also induced phenylamide accumulation. The compositions of the induced amides varied depending on the plant hormone used, and cinnamoyltryptamine, cinnamoylserotonin, and cinnamoyltyramine were not induced by the plant hormones. These findings suggest that several plant hormones and additional factors are involved in phenylamide accumulation in response to pathogen infection in rice.     

Crystal structure of bacteriophage ϕNIT1 zinc peptidase PghP that hydrolyzes γ-glutamyl linkage of bacterial poly-γ-glutamate

Poly‐γ‐glutamate hydrolase P (PghP) of Bacillus subtilis bacteriophage ΦNIT1 hydrolyzes the γ‐glutamyl peptide linkage of extracellular poly‐γ‐glutamate produced by bacilli, which facilitates infection and propagation of phage progenies. Crystal structure of PghP was determined at a resolution of 1.9 Å. Structure of PghP was elucidated as a globular protein with an open α/β mixed core structure and a seven‐stranded parallel/anti‐parallel β‐sheet. The β‐sheet contained a core four‐stranded parallel β‐sheet. A zinc‐binding motif, His‐Glu‐His, was identified at the C‐terminal end of the β‐sheet. Structure analysis demonstrated that PghP, which had not been previously classified into any peptidase/protease family due to lack of amino acid sequence similarity with known enzymes, had a catalytic center containing a zinc ion and an overall topology resembling mammalian carboxypeptidase A and related enzymes. Structural comparisons indicated important amino acid residues of PghP for catalysis and recognition of the γ‐peptide bond of poly‐γ‐glutamate, which was confirmed by site‐directed mutagenesis of PghP. Proteins 2011. © 2012 Wiley Periodicals, Inc.     

Crystal structure and nucleic acid‐binding activity of the CRISPR‐associated protein Csx1 of Pyrococcus furiosus

In many prokaryotic organisms, chromosomal loci known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR‐associated (CAS) genes comprise an acquired immune defense system against invading phages and plasmids. Although many different Cas protein families have been identified, the exact biochemical functions of most of their constituents remain to be determined. In this study, we report the crystal structure of PF1127, a Cas protein of Pyrococcus furiosus DSM 3638 that is composed of 480 amino acids and belongs to the Csx1 family. The C‐terminal domain of PF1127 has a unique β‐hairpin structure that protrudes out of an α‐helix and contains several positively charged residues. We demonstrate that PF1127 binds double‐stranded DNA and RNA and that this activity requires an intact β‐hairpin and involve the homodimerization of the protein. In contrast, another Csx1 protein from Sulfolobus solfataricus P2 that is composed of 377 amino acids does not have the β‐hairpin structure and exhibits no DNA‐binding properties under the same experimental conditions. Notably, the C‐terminal domain of these two Csx1 proteins is greatly diversified, in contrast to the conserved N‐terminal domain, which appears to play a common role in the homodimerization of the protein. Thus, although P. furiosus Csx1 is identified as a nucleic acid‐binding protein, other Csx1 proteins are predicted to exhibit different individual biochemical activities. Proteins 2013. © 2012 Wiley Periodicals, Inc.