CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria.

Most cyanobacteria use a single type of cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer cyanophycin. The genomes of many N2-fixing cyanobacteria contain an additional gene that encodes a second type of cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide ß-aspartyl-arginine to form cyanophycin. Thus, CphA2 represents a novel type of cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N2-fixing conditions. Electron micrographs revealed that the mutant had fewer cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.

Identification of the light-independent phosphoserine pathway as an additional source of serine in the cyanobacterium Synechocystis sp. PCC 6803.

L-serine is one of the proteinogenic amino acids and participates in several essential processes in all organisms. In plants, the light-dependent photorespiratory and the light-independent phosphoserine pathways contribute to serine biosynthesis. In cyanobacteria, the light-dependent photorespiratory pathway for serine synthesis is well characterized, but the phosphoserine pathway has not been identified. Here, we investigated three candidate genes for enzymes of the phosphoserine pathway in Synechocystis sp. PCC 6803. Only the gene for the D-3-phosphoglycerate dehydrogenase is correctly annotated in the genome database, whereas the 3-phosphoserine transaminase and 3-phosphoserine phosphatase (PSP) proteins are incorrectly annotated and were identified here. All enzymes were obtained as recombinant proteins and showed the activities necessary to catalyse the three-step phosphoserine pathway. The genes coding for the phosphoserine pathway were found in most cyanobacterial genomes listed in CyanoBase. The pathway seems to be essential for cyanobacteria, because it was impossible to mutate the gene coding for PSP in Synechocystis sp. PCC 6803 or in Synechococcus elongatus PCC 7942. A model approach indicates a 30-60% contribution of the phosphoserine pathway to the overall serine pool. Hence, this study verified that cyanobacteria, similar to plants, use the phosphoserine pathway in addition to photorespiration for serine biosynthesis.

All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120.

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis-Menten kinetics, with kcat = 48.2 s(-1) at pH 7.5 and 28 °C and KM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the 'bi-bi ping-pong mechanism'. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

From interactions of single transmembrane helices to folding of alpha-helical membrane proteins: analyzing transmembrane helix-helix interactions in bacteria.

Despite a wide variety of biological functions, alpha-helical membrane proteins display a rather simple transmembrane architecture. Although not many high resolution structures of transmembrane proteins are available today, our understanding of membrane protein folding has emerged in the recent years. Now we begin to develop a basic understanding of the forces that guide folding and interaction of alpha-helical membrane proteins. Some structural requirements for transmembrane helix interactions are defined, and common motifs have been discovered in the recent years which can drive helix-helix interactions. Nevertheless, many open questions remain to be addressed in future studies. One general problem with investigating transmembrane helix interactions is the limited number of appropriate tools, which can be applied to investigate membrane protein folding. Only recently several new techniques have been developed and established, including genetic systems, which allow measuring transmembrane helix interactions in vitro and in vivo. In the first part of this review, we summarize several aspects of the current understanding of membrane protein folding and assembly. In the second part, we discuss genetic systems, which were developed in the recent years to measure interaction of transmembrane helices in the inner membrane of E. coli.

PetG and PetN, but not PetL, are essential subunits of the cytochrome b6f complex from Synechocystis PCC 6803.

The cytochrome b(6)f complex consists of four large core subunits and an additional four low molecular weight subunits, the function of which is elusive thus far. Here we sought to determine whether small subunits PetG, PetL, and PetN are essential for a cyanobacterial cytochrome b(6)f complex. We found that only PetL is dispensable, whereas PetG and PetN appear to be essential. Possible roles of the small cytochrome b(6)f complex subunits are discussed, and observations from our study are compared with previous findings.

Ssr2998 of Synechocystis sp. PCC 6803 is involved in regulation of cyanobacterial electron transport and associated with the cytochrome b6f complex.

To analyze the function of a protein encoded by the open reading frame ssr2998 in Synechocystis sp. PCC 6803, the corresponding gene was disrupted, and the generated mutant strain was analyzed. Loss of the 7.2-kDa protein severely reduced the growth of Synechocystis, especially under high light conditions, and appeared to impair the function of the cytochrome b6 f complex. This resulted in slower electron donation to cytochrome f and photosystem 1 and, concomitantly, over-reduction of the plastoquinone pool, which in turn had an impact on the photosystem 1 to photosystem 2 stoichiometry and state transition. Furthermore, a 7.2-kDa protein, encoded by the open reading frame ssr2998, was co-isolated with the cytochrome b6 f complex from the cyanobacterium Synechocystis sp. PCC 6803. ssr2998 seems to be structurally and functionally associated with the cytochrome b6 f complex from Synechocystis, and the protein could be involved in regulation of electron transfer processes in Synechocystis sp. PCC 6803.

Assembly of a transmembrane b-type cytochrome is mainly driven by transmembrane helix interactions.

Folding, assembly and stability of alpha-helical membrane proteins is still not very well understood. Several of these membrane proteins contain cofactors, which are essential for their function and which can be involved in protein assembly and/or stabilization. The effect of heme binding on the assembly and stability of the transmembrane b-type cytochrome b'559 was studied by fluorescence resonance energy transfer. Cytochrome b'559 consists of two monomers of a 44 amino acid long polypeptide, which contains one transmembrane domain. The synthesis of two variants of the b'559 monomer, each carrying a specific fluorescent dye, allowed monitoring helix-helix interactions in micelles by resonance energy transfer. The measurements demonstrate that the transmembrane peptides dimerize in detergent in the absence and presence of the heme cofactor. Cofactor binding only marginally enhances dimerization and, apparently, the redox state of the heme group has no effect on dimerization.

The stability of transmembrane helix interactions measured in a biological membrane.

Despite some promising progress in the understanding of membrane protein folding and assembly, there is little experimental information regarding the thermodynamic stability of transmembrane helix interactions and even less on the stability of transmembrane helix-helix interactions in a biological membrane. Here we describe an approach that allows quantitative measurement of transmembrane helix interactions in a biological membrane, and calculation of changes in the interaction free energy resulting from substitution of single amino acids. Dimerization of several variants of the glycophorin A transmembrane domain are characterized and compared to the wild-type (wt) glycophorin A transmembrane helix dimerization. The calculated DeltaDeltaG(app) values are further compared with values found in the literature. In addition, we compare interactions between the wt glycophorin A transmembrane domain and helices in which critical glycine residues are replaced by alanine or serine, respectively. The data demonstrate that replacement of the glycine residues by serine is less destabilizing than replacement by alanine with a DeltaDeltaG(app) value of about 0.4 kcal/mol. Our study comprises the first measurement of a transmembrane helix interaction in a biological membrane, and we are optimistic that it can be further developed and applied.

Defining the structural basis for assembly of a transmembrane cytochrome.

To define the structural basis for cofactor binding to membrane proteins, we introduce a manageable model system, which allows us, for the first time, to study the influence of individual transmembrane helices and of single amino acid residues on the assembly of a transmembrane cytochrome. In vivo as well as in vitro analyses indicate central roles of single amino acid residues for either interaction of the transmembrane helices or for binding of the cofactor. The results clearly show that interaction of the PsbF transmembrane helix is independent from binding of the heme cofactor. On the other hand, binding of the cofactor highly depends on helix-helix interactions. By site-directed mutagenesis critical amino acid residues were identified, which are involved in the assembly of a functional transmembrane cytochrome. Especially, a highly conserved glycine residue is critical for interaction of the transmembrane helices and assembly of the cytochrome. Based on the two-stage-model of alpha-helical membrane protein folding, the presented results clearly indicate a third stage of membrane protein folding, in which a cofactor binds to a pre-assembled transmembrane protein.

PetC1 is the major Rieske iron-sulfur protein in the cytochrome b6f complex of Synechocystis sp. PCC 6803.

Many of the completely sequenced cyanobacterial genomes contain a gene family that encodes for putative Rieske iron-sulfur proteins. The Rieske protein is one of the large subunits of the cytochrome bc-type complexes involved in respiratory and photosynthetic electron transfer. In contrast to all other subunits of this complex that are encoded by single genes, the genome of the cyanobacterium Synechocystis PCC 6803 contains three petC genes, all encoding potential Rieske subunits. Most interestingly, any of the petC genes can be deleted individually without altering the Synechocystis phenotype dramatically. In contrast, double deletion experiments revealed that petC1 and petC2 cannot be deleted in combination, whereas petC3 can be deleted together with any of the other two petC genes. Further results suggest a different physiological function for each of the Rieske proteins. Whereas PetC2 can partly replace the dominating Rieske isoform PetC1, PetC3 is unable to functionally replace either PetC1 or PetC2 and may have a special function involving a special donor with a lower redox potential than plastoquinone. A predominant role of PetC1, which is (partly) different from PetC2, is suggested by the mutational analysis and a detailed characterization of the electron transfer reactions in the mutant strains.