Global proteome response of Synechocystis 6803 to extreme copper environments applied to control the activity of the inducible petJ promoter.

AIMS: Cyanobacteria are prokaryotes performing oxygenic photosynthesis, and they can be engineered to harness solar energy for production of commodity and high-value chemicals by means of synthetic biology. The Cu2+ -regulated petJ promoter (PpetJ ), which controls the expression of the endogenous cytochrome c553, can be used for expression of foreign products in Synechocystis 6803. We aimed to disclose potential bottlenecks in application of the PpetJ in synthetic biology approaches. METHODS AND RESULTS: Quantitative label-free mass spectrometry revealed global proteome changes which occurred during nutrient conditions which repress or activate of PpetJ in Synechocystis 6803. CONCLUSIONS: Some irreversible proteome alterations were discovered due to the copper stress, including downregulation of the ribosomal proteins, significant changes in protein amounts of the cell surface layer and the outer and inner membranes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed limitations in the use of PpetJ for biotechnological applications.

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes.

Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition-repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

Truncated aspartate aminotransferase from alkalophilic Bacillus circulans with deletion of N-terminal 32 amino acids is a non-functional monomer in a partially structured state.

Aspartate aminotransferase (AspAT) from alkalophilic Bacillus circulans contains an additional N-terminal sequence of 32 amino acid residues that are absent in all other AspATs from different sources. Modeling suggested that this sequence forms two alpha-helical segments which establish a continuous network of interactions on the surface of the molecule. In the present study, we studied the role of the N-terminal sequence in folding and stability of AspAT by applying the scanning calorimetry, and CD and fluorescence spectroscopies to the native and truncated enzymes. Truncated AspAT (Delta2alpha mutant) devoid of N-terminal residues cannot provide sufficient potential of quaternary intersubunit and subunit-cofactor interactions, which results in a monomeric non-functional conformation. However, the residual tertiary interactions in the Delta2alpha mutant are sufficient to: i) provide stability of a residual structure over a wide pH range; ii) confer moderate cooperativity of the denaturant-induced transition while only low cooperativity of the thermal transition, and iii) maintain the hydrophobic core of a part of the structure which prevents aromatic fluorophores from quenching by water. Furthermore, the present study provides evidence that AspAT from the alkalophilic bacterium follows unfolding pathway comprising a stable non-functional intermediate, in contrast to a two-state mechanism of the thermophilic AspAT from Sulfolobus solfataricus.

Aspartate aminotransferase from an alkalophilic Bacillus contains an additional 20-amino acid extension at its functionally important N-terminus.

Aspartate aminotransferase (AspAT), responsible for a minor part of the total AspAT enzymic activity in alkalophilic Bacillus circulans, was purified, its N-terminal amino acid sequence was determined, and its gene was cloned as two separate fragments. DNA sequencing showed an open reading frame of 432 amino acids (M(r) 47,439) exhibiting moderately low homology with AspATs from other sources. Sequence alignment of the enzyme with chicken mitochondrial, chicken cytoplasmic and Escherichia coli AspATs was performed with the MULTALIN program and further optimized assuming that the three-dimensional structures of the proteins were conserved. The primary structure of the studied AspAT diverged markedly from the others in the catalytically important small domain and in a segment of 31 amino acids in the large domain. The functional N-terminal arm was about two times longer than those of AspATs from other sources. According to the molecular model, the unique regions of B. circulans AspAT are all located together, forming a continuous network of contacts. Additional contacts formed by the elongated N-terminal arm may result in some limitation of domain movements in the alkalophilic enzyme in comparison to in other known AspATs.

Crystallization and preliminary X-ray analysis of phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus.

Recombinant phosphoserine aminotransferase (EC 2.6.1.52) from Bacillus circulans subsp. alkalophilus was crystallized at room temperature from 0.1 M sodium acetate buffer, pH 4.6, and 2% PEG 20000, using macroseeding techniques. The crystals diffract X-rays to at least 2.0 A nominal resolution. They belong to space group C2 with unit cell dimensions a = 93.2 A, b = 93.1 A, c = 45.6 A, alpha = 90.0 degrees, beta = 106.8 degrees, gamma = 90.0 degrees. A native data set to 2.3 A has been collected. Assuming an average packing density of the crystals, there is one monomer in the asymmetric unit, resulting in a calculated solvent content of 48.2%.

Phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus: purification, gene cloning and sequencing.

Two peaks of aspartate aminotransferase (AspAT) catalytic activity were observed during DEAE chromatography of a protein extract from alkalophilic B. circulans. The enzyme purified from the major peak appeared to be not aspartate but phosphoserine aminotransferase (PSAT) with a considerably high AspAT side activity. The sequence of the enzyme N-terminus was determined, and the PSAT gene was cloned as two separate fragments. DNA sequencing revealed the open reading frame for the PSAT starting from TTG, putative ribosomal binding site and terminator of transcription. The PSAT gene encodes a protein of 361 amino acids (M(r) 39793) which shows moderate homology to other known phosphoserine aminotransferases (36-46% of identity, 60-64% of similarity). The PSAT from the alkalophile shares with all of them the consensus sequence pattern around the pyridoxal 5'-phosphate attachment site.

The role of histidine residues in the catalytic act of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Our previous study on cyclomaltodextrin glucanotransferase (CGTase) by chemical modification implied the importance of one or two histidine residues in the cyclization reaction of the enzyme. Based on a computer modelled three-dimensional structure of the CGTase, five histidine residues were chosen as targets for the site-directed mutagenesis. The histidine residues 98, 140, 233 and 327 were replaced by aspartate and His-177 by proline using polymerase chain reaction-mediated techniques. The CGTase variants H98D, H140D, H233D and H327D resulted in a profound decrease in the cyclizing and amylolytic activities, while mutation H177P had little influence on the activities but affected the thermal stability and the width of the pH optimum. It is suggested that His-98 functions as (or as a significant part of) the subsite 2 for the binding of the substrate in CGTase and therefore H98D destabilizes the intermediate for cyclization, but does not markedly affect the hydrolytic reactions. Mutants H140D and H233D produced only minor amounts of alpha-cyclodextrin, did not exhibit substrate inhibition with maltotriose and showed non-Michaelis-Menten kinetics. It is proposed that the variants H140D, H233D and H327D cause steric hindrances near the active center, while mutation H177D has similar consequences on the same site spatially.