Markus Piotrowski

A giant chlorophyll–protein complex induced by iron deficiency in cyanobacteria

Cyanobacteria are abundant throughout most of the worlds water bodies and contribute significantly to global primary productivity through oxygenic photosynthesis. This reaction is catalysed by two membrane-bound protein complexes, photosystem I (PSI) and photosystem II (PSII), which both contain chlorophyll-binding subunits functioning as an internal antenna. In addition, phycobilisomes act as peripheral antenna systems, but no additional light-harvesting systems have been found under normal growth conditions. Iron deficiency, which is often the limiting factor for cyanobacterial growth in aquatic ecosystems, leads to the induction of additional proteins such as IsiA (ref. 3). Although IsiA has been implicated in chlorophyll storage, energy absorption and protection against excessive light, its precise molecular function and association to other proteins is unknown. Here we report the purification of a specific PSI–IsiA supercomplex, which is abundant under conditions of iron limitation. Electron microscopy shows that this supercomplex consists of trimeric PSI surrounded by a closed ring of 18 IsiA proteins binding around 180 chlorophyll molecules. We provide a structural characterization of an additional chlorophyll-containing, membrane-integral antenna in a cyanobacterial photosystem.

Phosphorylation of Thr-948 at the C Terminus of the Plasma Membrane H+-ATPase Creates a Binding Site for the Regulatory 14-3-3 Protein

The plant plasma membrane H+-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H+-ATPase–14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT948V, at the C terminus of the H+-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H+-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H+-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H+-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H+-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H+-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H+-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H+-ATPase in vivo. Indeed, replacing Thr-948 in the plant H+-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H+-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H+-ATPase activity in the plant and thus for plant growth.

Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster

Abstract. Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5′→3′ direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 °C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 °C) and PPN (35–40 °C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of β-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification.

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,K m = 85 μm). 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 μm, 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 theLeCPK1 N terminus was found to be required for plasma membrane targeting.

Evolution of heteromeric nitrilase complexes in Poaceae with new functions in nitrile metabolism

Members of the nitrilase 4 (NIT4) family of higher plants catalyze the conversion of β-cyanoalanine to aspartic acid and asparagine, a key step in cyanide detoxification. Grasses (Poaceae) possess two different NIT4 homologs (NIT4A and NIT4B), but none of the recombinant Poaceae enzymes analyzed showed activity with β-cyanoalanine, whereas protein extracts of the same plants clearly posses this activity. Sorghum bicolor contains three NIT4 isoforms SbNIT4A, SbNIT4B1, and SbNIT4B2. Individually, each isoform does not possess enzymatic activity whereas the heteromeric complexes SbNIT4A/B1 and SbNIT4A/B2 hydrolyze β-cyanoalanine with high activity. In addition, the SbNIT4A/B2 complex accepts additional substrates, the best being 4-hydroxyphenylacetonitrile. Corresponding NIT4A and NIT4B isoforms from other Poaceae species can functionally complement the sorghum isoforms in these complexes. Site-specific mutagenesis of the active site cysteine residue demonstrates that hydrolysis of β-cyanoalanine is catalyzed by the NIT4A isoform in both complexes whereas hydrolysis of 4-hydroxyphenylacetonitrile occurs at the NIT4B2 isoform. 4-Hydroxyphenylacetonitrile was shown to be an in vitro breakdown product of the cyanogenic glycoside dhurrin, a main constituent in S. bicolor. The results indicate that the SbNIT4A/B2 heterocomplex plays a key role in an endogenous turnover of dhurrin proceeding via 4-hydroxyphenylacetonitrile and thereby avoiding release of toxic hydrogen cyanide. The operation of this pathway would enable plants to use cyanogenic glycosides as transportable and remobilizable nitrogenous storage compounds. Through combinatorial biochemistry and neofunctionalizations, the small family of nitrilases has gained diverse biological functions in nitrile metabolism.

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 β-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.

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.

Synthesis of the D2 Protein of Photosystem II in Chlamydomonas Is Controlled by a High Molecular Mass Complex Containing the RNA Stabilization Factor Nac2 and the Translational Activator RBP40

Gene expression in chloroplasts is regulated mainly at the posttranscriptional level. In the green alga Chlamydomonas reinhardtii, synthesis of the D2 protein (PsbD), which is the rate-determining subunit for the assembly of photosystem II, depends on the RNA stability factor Nac2. In addition, the RNA binding protein RBP40 has been implicated in translational control via a U-rich element in the 5′ untranslated region (5′UTR) of the psbD mRNA. Here, we report the identification of the RBP40 gene based on mass spectrometric analysis of its purified product. Unexpectedly, this was found to be identical to the previously described RNA binding protein RB38, which had been suggested to be involved in the regulation of D1 protein synthesis. However, we show that RBP40 binds to the psbD 5′UTR in a Nac2-dependent fashion both in vitro and in vivo. Molecular characterization of RBP40 RNA interference lines confirmed that RBP40 specifically affects the initiation of D2 synthesis. Native polyacrylamide gel electrophoresis, coimmunoprecipitation, and sedimentation analyses revealed that Nac2 and RBP40 form parts of a complex of 550 kD that is displaced from the psbD mRNA prior to polysome assembly. Together, these data indicate that the processes of 5′UTR-mediated RNA stabilization and translation initiation are tightly coupled in Chlamydomonas.

Accumulation of Pathogenesis-Related Proteins in the Apoplast of a Susceptible Cultivar of Apple (Malus domestica cv. Elstar) after Infection by Venturia inaequalis and Constitutive Expression of PR Genes in the Resistant Cultivar Remo

Leaves of apple (Malus domestica cv. Elstar) were infected with a cloned isolate of the apple scab Venturia inaequalis. The intercellular washing fluid (IWF) of these plants was collected and the variation in the composition of proteins in the IWF was analysed by SDS-PAGE and two-dimensional gel electrophoresis during and after the infection with V. inaequalis, the causal agent of apple scab. The subsequent analysis of induced proteins by electron spray ionization quadrupole time of flight mass spectroscopy revealed the presence of β-1,3-glucanase, chitinase, thaumatin-like protein and a cysteine-like protease in M. domestica leaves infected by V. inaequalis. These results were confirmed by immunoblotting with antibodies against some of these proteins. Moreover, a non-specific lipid transfer protein was identified in uninfected leaves: the amount declined to a non-detectable level within the first week after infection by V. inaequalis. The analysis of the IWF of M. domestica cv. Remo, bearing resistances to apple scab, powdery mildew and fire blight, showed a protein pattern comparable to that of the IWF from V. inaequalis infected leaves from cultivar Elstar indicating the constitutive production at least of some of the pathogenesis-related proteins in the resistant cultivar.

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 β-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.