Biomedical subjects

Markus Piotrowski

Publications and source records attributed to Markus Piotrowski.

At least 19 recordsLinked to original sources

PubMed2006

Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway.

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+-binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses.

Amidohydrolases↗

PubMed2006

Multiple layers of temporal and spatial control regulate accumulation of the fruiting body-specific protein APP in Sordaria macrospora and Neurospora crassa.

During fungal fruiting body development, specialized cell types differentiate from vegetative mycelium. We have isolated a protein from the ascomycete Sordaria macrospora that is not present during vegetative growth but accumulates in perithecia. The protein was sequenced by mass spectrometry and the corresponding gene was termed app (abundant perithecial protein). app transcript occurs only after the onset of sexual development; however, the formation of ascospores is not a prerequisite for APP accumulation. The transcript of the Neurospora crassa ortholog is present prior to fertilization, but the protein accumulates only after fertilization. In crosses of N. crassa Deltaapp strains with the wild type, APP accumulates when the wild type serves as female parent, but not in the reciprocal cross; thus, the presence of a functional female app allele is necessary and sufficient for APP accumulation. These findings highlight multiple layers of temporal and spatial control of gene expression during fungal development.

Alleles↗

PubMed2006

Chloroplast heat shock protein Cpn60 from Chlamydomonas reinhardtii exhibits a novel function as a group II intron-specific RNA-binding protein.

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RNA. Using FPLC chromatography of chloroplast protein extracts, a 61-kDa RNA-binding protein was isolated and then tentatively identified by mass spectrometry as the chloroplast heat shock protein Cpn60. Heterologous Cpn60 protein was used in RNA protein gel mobility shift assays and revealed that the ATPase domains of Cpn60 mediates the specific binding of two group II intron RNAs, derived from the homologous chloroplast psaA gene and the heterologous mitochondrial LSU rRNA gene. The function of Cpn60 as a general organelle splicing factor is discussed.

Animals↗

PubMed2006

Cyanide metabolism in higher plants: cyanoalanine hydratase is a NIT4 homolog.

Cyanoalanine hydratase (E.C. 4.2.1.65) is an enzyme involved in the cyanide detoxification pathway of higher plants and catalyzes the hydrolysis of beta-cyano-L-alanine to asparagine. We have isolated the enzyme from seedlings of blue lupine (Lupinus angustifolius) to obtain protein sequence information for molecular cloning. In contrast to earlier reports, extracts of blue lupine cotyledons were found also to contain cyanoalanine-nitrilase (E.C. 3.5.5.4) activity, resulting in aspartic acid production. Both activities co-elute during isolation of cyanoalanine hydratase and are co-precipitated by an antibody directed against Arabidopsis thaliana nitrilase 4 (NIT4). The isolated cyanoalanine hydratase was sequenced by nanospray-MS/MS and shown to be a homolog of Arabidopsis thaliana and Nicotiana tabacum NIT4. Full-length cDNA sequences for two NIT4 homologs from blue lupine were obtained by PCR using degenerate primers and RACE-experiments. The recombinant LaNIT4 enzymes, like Arabidopsis NIT4, hydrolyze cyanoalanine to asparagine and aspartic acid but show a much higher cyanoalanine-hydratase activity. The two nitrilase genes displayed differential but overlapping expression. Taken together these data show that the so-called 'cyanoalanine hydratase' of plants is not a bacterial type nitrile hydratase enzyme but a nitrilase enzyme which can have a remarkably high nitrile-hydratase activity.

Alanine↗

PubMed2005

Rubrerythrin and peroxiredoxin: two novel putative peroxidases in the hydrogenosomes of the microaerophilic protozoon Trichomonas vaginalis.

The parasitic flagellate Trichomonas vaginalis contains hydrogenosomes, anaerobic organelles related to mitochondria, that generate ATP from the fermentative conversion of pyruvate to acetate, CO2 and molecular hydrogen. Although an essentially anaerobic organism, Trichomonas encounters low oxygen concentrations in its natural habitat and has to protect itself, and especially the oxygen-sensitve enzymes of hydrogenosomal metabolism, from oxidative damage. We have identified two novel proteins in the hydrogenosomal proteome with strong similarity to two putative prokaryotic peroxidases, rubrerythrin and periplasmic thiol peroxidase. Both proteins have previously been found in many prokaryotes but were not known from eukaryotes, suggesting a significant prokaryotic component in the oxygen-detoxification system of trichomonad hydrogenosomes.

Amino Acid Sequence↗

PubMed2004

Mitochondrial trans-2-enoyl-CoA reductase of wax ester fermentation from Euglena gracilis defines a new family of enzymes involved in lipid synthesis.

Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (km=68 microm) and trans-2-hexenoyl-CoA (km=91 microm). In the crotonyl-CoA-dependent reaction, both NADH (km=109 microm) or NADPH (km=119 microm) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoyl-CoA, to one member of this gene family of previously unknown function.

Adenosine Triphosphate↗

PubMed2004

Bacteriophage T4 alpha-glucosyltransferase: a novel interaction with gp45 and aspects of the catalytic mechanism.

The bacteriophage T4 alpha- and beta-glucosyltransferases (AGT and BGT) catalyse the transfer of glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine of T4 DNA in an alpha- and beta-conformation, respectively. Following the 3D structure of BGT and a secondary structure alignment of AGT and BGT, we performed a site-directed mutagenesis of AGT. A two-domain structure was deduced, with an open substrate-free and a closed substrate-bound conformation. We also identified specific amino acids involved in DNA binding. The identification of a protein-protein interaction of AGT and gp45 which is a part of the T4 replication complex supports the idea that T4 DNA is alpha-glucosylated immediately after synthesis. BGT then glucosylates those hydroxymethyl cytosines not previously served by AGT.

Amino Acid Sequence↗

PubMed2004

Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure.

The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.

Amino Acids↗

PubMed2004

Investigations on epiphytic living Pseudomonas species from Malus domestica with an antagonistic effect to Venturia inaequalis on isolated plant cuticle membranes.

In order to understand better the survival and mutual interaction of epiphytic bacteria and fungi on apple plants, bacteria collected from these plants were cultivated on intact adaxial, stoma free cuticle membranes originally obtained from apple. The bacteria were labelled with luciferase genes from Vibrio harveyi in order to follow up their development and activity on the isolated cuticles. Our finding was that the epiphytic bacteria can have access to nutrients below the cuticle without causing damage to these cuticular membranes. Bacterial proteins may enable this nutrient mobilization and we found, indeed, that more than 46 proteins that must have been delivered by the bacteria in response to interaction with the cuticles as they could be found below the cuticle membrane. Eight major representatives of this group of external proteins have been sequenced with electron spray quadrupole time of flight mass spectrometry and subsequently identified by data base homology search as a flagellin, a porin type protein and proteins that are involved in amino acid recruitment and metabolism.

Antibiosis↗

PubMed2003

The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid.

We isolated two nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward beta-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). ZmNIT2 converts IAN to IAA at least seven to 20 times more efficiently than AtNIT1/2/3. Quantitative real-time polymerase chain reaction revealed the gene expression of both nitrilases in maize kernels where high concentrations of IAA are synthesized tryptophan dependently. Nitrilase protein and endogenous nitrilase activity are present in maize kernels together with the substrate IAN. These results suggest a role for ZmNIT2 in auxin biosynthesis.

Aminohydrolases↗

PubMed2003

Identification and characterization of plant agmatine iminohydrolase, the last missing link in polyamine biosynthesis of plants.

The cloning, expression and characterization of plant agmatine iminohydrolase (AIH, also known as agmatine deiminase, EC 3.5.3.12) is described. Recombinant AIH of Arabidopsis thaliana forms dimers and catalyzes the specific conversion of agmatine to N-carbamoylputrescine and ammonia. Biochemical data suggested that cysteine side chains are involved in catalysis. However, site-directed mutagenesis of the two highly conserved cysteine residues of AIH showed that these cysteines are important but not essential for activity, arguing against a thioester substrate-enzyme intermediate during catalysis. This work represents the completion of the cloning of the arginine decarboxylase pathway genes of higher plants.

Ammonia↗

PubMed2003

Physiological and biochemical characterization of ethylene-generated gravicompetence in primary shoots of coleoptile-less gravi-incompetent rye seedlings.

A recent study demonstrated that gravi-incompetent coleoptile-less seedlings of rye exhibit gravi-competence after exogenous application of ethylene. Treatments and conditions which induce and interfere with this phenomenon were analysed in more detail. Aminocyclopropane-1-carboxylic acid (ACC) as a precursor of ethylene has similar gravicompetence-inducing effects and also appropriate conditions of light, which strongly enhances ethylene synthesis. Both effects can be inhibited by the ethylene-perception blocking agent methylcyclopropene (MCP) or inhibitors of ethylene synthesis such as aminovinylglycine (AVG), indicating that light exerts its gravicompetence-generating effect via induced/enhanced ethylene synthesis. Gain in gravicompetence is accompanied by the induced/enhanced occurrence of calreticulin and lipoxygenase as detected by 2D-gels and Q-TOFF-analyses. Previously gravicompetent, light-grown coleoptile-less seedlings are characterized by gravi-incompetent growth during subsequent horizontal gravistimulation when perception of ethylene is inhibited by MCP. The results demonstrate that continuous perception of ethylene is an indispensable step, permanently required for the regulation of gravitropic growth in germinating primary shoots of rye, either within the process of graviperception and/or of the transduction of the gravi-signal.

Amino Acid Sequence↗

PubMed2002

Plant C-N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis.

A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His(6)-tagged protein and purified to apparent homogeneity by Ni(2+)-chelate affinity chromatography. The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria. N-carbamoylputrescine amidohydrolase activity was confirmed by identification of two of the three occurring products, namely putrescine and ammonia. In contrast, no enzymatic activity could be detected when applying various compounds including nitriles, amines, and amides as well as other N-carbamoyl compounds, indicating the specificity of the enzyme for N-carbamoylputrescine. Like the homologous beta-alanine synthases, NLP1 showed positive cooperativity toward its substrate. The native enzyme had a molecular mass of 279 kDa as shown by blue-native polyacrylamide gel electrophoresis, indicating a complex of eight monomers. Expression of the NLP1 gene was found in all organs investigated, but it was not induced upon osmotic stress, which is known to induce biosynthesis of putrescine. This is the first report of cloning and expression of a plant N-carbamoylputrescine amidohydrolase and the first time that N-carbamoylputrescine amidohydrolase activity of a recombinant protein could be shown in vitro. NLP1 is one of the two missing links in the arginine decarboxylase pathway of putrescine biosynthesis in higher plants.

Arabidopsis↗

PubMed2002

Occurrence and formation of indole-3-acetamide in Arabidopsis thaliana.

An HPLC/GC-MS/MS technique (high-pressure liquid chromatography in combination with gas chromatography-tandem mass spectrometry) has been worked out to analyze indole-3-acetamide (IAM) with very high sensitivity, using isotopically labelled IAM as an internal standard. Using this technique, the occurrence of IAM in sterile-grown Arabidopsis thaliana (L.) Heynh. was demonstrated unequivocally. In comparison, plants grown under non-sterile conditions in soil in a greenhouse showed approximately 50% higher average levels of IAM, but the differences were not statistically significant. Thus, microbial contributions to the IAM extracted from the tissue are likely to be minor. Levels of IAM in sterile-grown seedlings were highest in imbibed seeds and then sharply declined during the first 24 h of germination and further during early seedling development to remain below 20-30 pmol g(-1) fresh weight throughout the rosette stage. The decline in indole-3-aetic acid (IAA) levels during germination was paralleled by a similar decline in IAM levels. Recombinant nitrilase isoforms 1, 2 and 3, known to synthesize IAA from indole-3-acetonitrile, were shown to produce significant amounts of IAM in vitro as a second end product of the reaction besides IAA. NIT2 was earlier shown to be highly expressed in developing and in mature A. thaliana embryos, and NIT3 is the dominantly active gene in the hypocotyl and the cotyledons of young, germinating seedlings. Collectively, these data suggest that the elevated levels of IAM in seeds and germinating seedlings result from nitrilase action on indole-3-acetonitrile, a metabolite produced in the plants presumably from glucobrassicin turnover.

Aminohydrolases↗

PubMed2002

A role for nitrilase 3 in the regulation of root morphology in sulphur-starving Arabidopsis thaliana.

Arabidopsis thaliana expresses four nitrilases, three of which (NIT1, NIT2 and NIT3) are able to convert indole-3-acetonitrile to indole-3-acetic acid (IAA), the plant growth hormone, while the isozyme NIT4 is a beta-cyano-l-alanine hydratase/nitrilase. NIT3 promoter activity is marginal in leaves or roots of vegetative plants and undetectable in bolting and flowering plants, but its level increases strongly when plants experience sulphur deprivation. No other nitrilase genes respond to sulphur supply/deficiency. Neither N- nor P-deprivation cause detectable changes in NIT3 promoter activity. In transgenic plants expressing uidA under the control of the NIT3 promoter (NIT3p::uidA), sulphate deprivation leads to the appearance of beta-glucuronidase activity in shoots and particularly in roots, most strongly in the conductive tissues and lateral root primordia. Deletion analysis allowed localization of the sulphur-responsive element to a 317 bp segment of the NIT3 promoter encompassing nt -2151 to -1834 upstream of the transcriptional start point. Both nitrilase polypeptide and nitrilase activity were also induced by sulphur starvation. NIT3 promoter activity was strongly induced by O-acetylserine, suggesting that, as is the case with enzymes of sulphate assimilation, sulphate deficiency may be communicated to NIT3 via an increase in the level of the cysteine precursor, O-acetylserine. During sulphur deprivation, a preferential depletion of the pool of the indole-3-acetonitrile precursor glucobrassicin compared with that of total glucosinolates was noticed. In the absence of an external sulphate supply, plants developed longer roots with a higher number of lateral roots. The increased growth of the root system occurred at the expense of shoot growth which was retarded under conditions of sulphur starvation. Taken together, these results suggest that a regulatory loop appears to exist by which sulphate deficiency, through an increase in glucobrassicin turnover and nitrilase 3 accumulation, initiates the production of extra auxin leading to increased root growth and branching, thus allowing the root system to penetrate new areas of soil effectively to gain access to fresh supplies of sulphur.

Aminohydrolases↗

PubMed2002

LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization.

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 microM). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 microM, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting.

Amino Acid Sequence↗

PubMed2002

The plastid transcription kinase from mustard (Sinapis alba L.). A nuclear-encoded CK2-type chloroplast enzyme with redox-sensitive function.

The plastid transcription kinase (PTK), a component of the major RNA polymerase complex from mustard chloroplasts, has been implicated in redox-mediated regulation of plastid gene expression. A cloning strategy to define the PTK gene(s) resulted in the isolation of a full-length cDNA for a protein with overall high homology with the alpha subunit of cytosolic casein kinase (CK2) that contained an N-terminal extension for a putative plastid transit peptide. Using in organello chloroplast import studies, immunodetection and MS, we found that the corresponding protein, termed cpCK2alpha, is targeted to the chloroplast and is associated with the plastid RNA polymerase PEP-A. The bacterially overexpressed protein shows CK2 kinase activity and is subject to glutathione inhibition in the same way as authentic chloroplast PTK. Furthermore, it readily phosphorylates components of the plastid transcription apparatus in vitro with a substrate specificity similar to that of PTK.

Amino Acid Sequence↗

PubMedDate not supplied

Up-regulation of pathogenesis-related proteins in the apoplast of Malus domestica after application of a non-pathogenic bacterium.

The intercellular washing fluid (IWF) of Malus domestica cv. Holsteiner Cox before and after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the leaves was investigated in a comparative manner. SDS-PAGE in combination with ESI Q-ToF mass spectrometry, and homology search in relevant data bases revealed the highly up-regulated expression of several pathogenesis-related plant proteins in the apoplast of the leaves treated with P. fluorescens. These proteins were beta3-1,3-glucanase, chitinase, thaumatin-like protein, ribonuclease-like protein, and a hevein-like protein. Moreover, a 9 kDa non-specific lipid transfer protein was significantly reduced after the application of P. fluorescens. The possible relevance of a pre-treatment of apple cultivars with the non-pathogenic bacterium P. fluorescens Bk3, as an alternative method to the treatment with fungicides, for increasing the resistance of susceptible apple cultivars against an infection with the fungus Venturia inaequalis is discussed.

Amino Acid Sequence↗