Systematic Characterization of the ADP-Ribose Pyrophosphatase Family in the Cyanobacterium Synechocystis sp. Strain PCC 6803

ABSTRACT We have characterized four putative ADP-ribose pyrophosphatases Sll1054, Slr0920, Slr1134, and Slr1690 in the cyanobacterium Synechocystis sp. strain PCC 6803. Each of the recombinant proteins was overexpressed in Escherichia coli and purified. Sll1054 and Slr0920 hydrolyzed ADP-ribose specifically, while Slr1134 hydrolyzed not only ADP-ribose but also NADH and flavin adenine dinucleotide. By contrast, Slr1690 showed very low activity for ADP-ribose and had four substitutions of amino acids in the Nudix motif, indicating that Slr1690 is not an active ADP-ribose pyrophosphatase. However, the quadruple mutation of Slr1690, T73G/I88E/K92E/A94G, which replaced the mutated amino acids with those conserved in the Nudix motif, resulted in a significant (6.1 × 102-fold) increase in the k cat value. These results suggest that Slr1690 might have evolved from an active ADP-ribose pyrophosphatase. Functional and clustering analyses suggested that Sll1054 is a bacterial type, while the other three and Slr0787, which was characterized previously (Raffaelli et al., FEBS Lett. 444:222-226, 1999), are phylogenetically diverse types that originated from an archaeal Nudix protein via molecular evolutionary mechanisms, such as domain fusion and amino acid substitution.

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Current knowledge and recent advances in understanding metabolism of the model cyanobacterium Synechocystis sp. PCC 6803

Bioscience Reports ◽

10.1042/bsr20193325 ◽

2020 ◽

Vol 40 (4) ◽

Cited By ~ 5

Author(s):

Lauren A. Mills ◽

Alistair J. McCormick ◽

David J. Lea-Smith

Keyword(s):

Amino Acids ◽

Heterotrophic Bacteria ◽

Membrane Lipids ◽

Current Knowledge ◽

Oxygenic Photosynthesis ◽

Photosynthetic Organisms ◽

Physiological Processes ◽

Synechocystis Sp ◽

Pcc 6803

Abstract Cyanobacteria are key organisms in the global ecosystem, useful models for studying metabolic and physiological processes conserved in photosynthetic organisms, and potential renewable platforms for production of chemicals. Characterizing cyanobacterial metabolism and physiology is key to understanding their role in the environment and unlocking their potential for biotechnology applications. Many aspects of cyanobacterial biology differ from heterotrophic bacteria. For example, most cyanobacteria incorporate a series of internal thylakoid membranes where both oxygenic photosynthesis and respiration occur, while CO2 fixation takes place in specialized compartments termed carboxysomes. In this review, we provide a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in Synechocystis sp. PCC 6803 (Synechocystis) involved in biosynthesis of sugar-based metabolites, amino acids, nucleotides, lipids, cofactors, vitamins, isoprenoids, pigments and cell wall components, in addition to the proteins involved in metabolite transport. While some pathways are conserved between model cyanobacteria, such as Synechocystis, and model heterotrophic bacteria like Escherichia coli, many enzymes and/or pathways involved in the biosynthesis of key metabolites in cyanobacteria have not been completely characterized. These include pathways required for biosynthesis of chorismate and membrane lipids, nucleotides, several amino acids, vitamins and cofactors, and isoprenoids such as plastoquinone, carotenoids, and tocopherols. Moreover, our understanding of photorespiration, lipopolysaccharide assembly and transport, and degradation of lipids, sucrose, most vitamins and amino acids, and haem, is incomplete. We discuss tools that may aid our understanding of cyanobacterial metabolism, notably CyanoSource, a barcoded library of targeted Synechocystis mutants, which will significantly accelerate characterization of individual proteins.

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Cloning of the psbK gene from Synechocystis sp. PCC 6803 and characterization of photosystem II in mutants lacking PSII-K

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)99134-9 ◽

1991 ◽

Vol 266 (17) ◽

pp. 11111-11115

Author(s):

M. Ikeuchi ◽

B. Eggers ◽

G.Z. Shen ◽

A. Webber ◽

J.J. Yu ◽

...

Keyword(s):

Photosystem Ii ◽

Synechocystis Sp ◽

Pcc 6803

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Characterization of a Synechocystis sp. PCC 6803 double mutant lacking the CyanoP and Ycf48 proteins of Photosystem II

Photosynthesis Research ◽

10.1007/s11120-015-0122-0 ◽

2015 ◽

Vol 124 (2) ◽

pp. 217-229 ◽

Cited By ~ 8

Author(s):

Simon A. Jackson ◽

Julian J. Eaton-Rye

Keyword(s):

Photosystem Ii ◽

Double Mutant ◽

Synechocystis Sp ◽

Pcc 6803

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Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Biochemical Journal ◽

10.1042/bj20102131 ◽

2011 ◽

Vol 435 (3) ◽

pp. 733-742 ◽

Cited By ~ 20

Author(s):

Pitter F. Huesgen ◽

Helder Miranda ◽

XuanTam Lam ◽

Manuela Perthold ◽

Holger Schuhmann ◽

...

Keyword(s):

Serine Proteases ◽

Protein Quality ◽

Biochemical Characterization ◽

Pdz Domain ◽

Control Mechanisms ◽

Ph Optimum ◽

Periplasmic Proteins ◽

Synechocystis Sp ◽

Pcc 6803

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.

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