Biochemical Characterization of Glucosylglycerol-Phosphate Synthase of Synechocystis sp. Strain PCC 6803: Comparison of Crude, Purified, and Recombinant Enzymes

Hagemann, Martin; Effmert, Uta; Kerstan, Thomas; Schoor, Arne; Erdmann, Norbert

doi:10.1007/s002840010301

Cited by 35 publications

(24 citation statements)

References 26 publications

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“…The synthesis of GG in P. marina proceeds via a two-step pathway, as in M. burtonii, which is common to other sugarrelated compatible solutes, such as trehalose, sucrose, MG, and glucosylglycerol (9,10,14,18,22). In P. marina, gpgS and gpgP are contained in an operon-like structure in which we identified genes for a glycerate kinase (gK), a glucosyltransferase (gT), and a histidine kinase (PhoR).…”

Section: Discussionmentioning

confidence: 99%

Glucosylglycerate Biosynthesis in the Deepest Lineage of the Bacteria : Characterization of the Thermophilic Proteins GpgS and GpgP from Persephonella marina

2007

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The pathway for the synthesis of glucosylglycerate (GG) in the thermophilic bacterium Persephonella marina is proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (GPG) synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). The sequences of gpgS and gpgP from the cold-adapted bacterium Methanococcoides burtonii were used to identify the homologues in the genome of P. marina, which were separately cloned and overexpressed as His-tagged proteins in Escherichia coli. The recombinant GpgS protein of P. marina, unlike the homologue from M. burtonii, which was specific for GDP-glucose, catalyzed the synthesis of GPG from UDP-glucose, GDP-glucose, ADP-glucose, and TDP-glucose (in order of decreasing efficiency) and from D-3-phosphoglycerate, with maximal activity at 90°C. The recombinant GpgP protein, like the M. burtonii homologue, dephosphorylated GPG and mannosyl-3-phosphoglycerate (MPG) to GG and mannosylglycerate, respectively, yet at high temperatures the hydrolysis of GPG was more efficient than that of MPG. Gel filtration indicates that GpgS is a dimeric protein, while GpgP is monomeric. This is the first characterization of genes and enzymes for the synthesis of GG in a thermophile.The compatible solute ␣-glucosylglycerate (GG) has been identified in the cyanobacterium Agmenellum quadruplicatum strain PCC7002, in the archaeon Methanohalophilus portucalensis strain FDF-1, in a salt-sensitive mutant of Halomonas elongata, and in the ␥-proteobacterium Erwinia chrysanthemi strain 3937, where it behaves as a compatible solute during osmotic stress under nitrogen-limiting conditions (8,21,26,33). This compatible solute is chemically related to mannosylglycerate (MG), which is widespread in (hyper)thermophilic bacteria and archaea and has been shown to serve as a compatible solute under salt stress in several of these organisms (1,35,36). However, MG has also been encountered in marine red algae, and the genes for the synthesis of MG have been found in the mesophilic bacterium "Dehalococcoides ethenogenes" (proposed name) (16,25). On the other hand, the accumulation of GG had been detected only in mesophilic bacteria and archaea (8,21,26,33). However, GG was recently shown to accumulate in Persephonella marina (H. Santos, personal communication), a thermophilic, strictly chemolithoautotrophic, microaerophilic, hydrogen-oxidizing bacterium isolated from a deep-sea hydrothermal vent which is a member of the order Aquificales (20). This bacterium has a temperature range for growth of between 55 and 80°C (optimum at 73°C) and grows optimally in media containing 2.5% (wt/vol) NaCl (20). The identification of GG in this organism, where it may have a role in osmoadaptation, prompted us to examine the pathway for its synthesis.The biosynthetic pathway for the synthesis of GG in Methanococcoides burtonii proceeds via a two-step pathway involving glucosyl-3-phosphoglycerate synthase (GpgS), which catalyzes the conversion of GDP-glucose and D-3-phosphoglycerate (3-PGA) to glucosyl-3-phosphogl...

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Section: Discussionmentioning

confidence: 99%

Glucosylglycerate Biosynthesis in the Deepest Lineage of the Bacteria : Characterization of the Thermophilic Proteins GpgS and GpgP from Persephonella marina

2007

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“…The syntheses of the compatible solutes glucosylglycerol, galactosylglycerol, trehalose, and sucrose share, with the synthesis of MG, the formation of a phosphorylated intermediate (7,11,14,37). Despite this common feature, the MPGSs of T. thermophilus, Pyrococcus spp., A. pernix, and an uncultured crenarchaeote share insignificant sequence similarity with these osmolyte-phosphate synthases and were classified in a separate glycosyltransferase family, designated GT55 (http: //afmb.cnrs-mrs.fr/ϳcazy/CAZY/index.html).…”

Section: Discussionmentioning

confidence: 99%

“…strain PCC 6803, trehalose-phosphate synthase from Mycobacterium smegmatis, and sucrose-phosphate synthase from Synechocystis. These enzymes, like P. horikoshii MPGS, are active in the absence of cations, but Mg 2ϩ stimulates their activity (7,14,29). The absolute cation dependence of the bacterial MPGP for activity also represents the main difference between the bacterial and the archaeal enzyme.…”

Section: Discussionmentioning

confidence: 99%

The Bacterium Thermus thermophilus , Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate

Empadinhas

Albuquerque

Henne

et al. 2003

Appl Environ Microbiol

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The biosynthetic pathway for the synthesis of the compatible solute ␣-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of ␣-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90°C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95°C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn 2؉ was most effective for MPGS, while Mn 2؉ , Co 2؉ , Mg 2؉ , and to a lesser extent Ni 2؉ activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.The compatible solute mannosylglycerate (MG) was originally identified in red algae of the order Ceramiales (4) but has never been encountered in mesophilic bacteria or archaea. MG is, however, a common compatible solute of halotolerant or slightly halophilic thermophilic and hyperthermophilic prokaryotes. The organisms that accumulate MG include the slightly halophilic euryarchaeotes of the genera Pyrococcus and Thermococcus (22, 23), the crenarchaeote Aeropyrum pernix, and the species Archaeoglobus veneficus and Archaeoglobus profundus (12). Among thermophilic bacteria, MG has been identified in Thermus thermophilus, Rhodothermus marinus, and Rubrobacter xylanophilus (28, 33). The role of MG as a compatible solute under salt stress has been confirmed in several of these organisms, and there is increasing evidence that this organic solute also has a role in stabilizing proteins from thermal denaturation, since it is one of the most efficient thermoprotectants known in vitro (3, 30).The biosynthetic route for the synthesis of MG has been examined in Rhodothermus marinus and Pyrococcus horikoshii. The synthesis of MG proceeds via two alternate routes in R. marinus. In one pathway, GDP-mannose is condensed with D-glycerate to produce MG in a single glycosyl transfer reaction catalyzed by MG synthase (26). In the other pathway, mannosyl-3-phosphoglycerate...

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“…This is based on the horizontal transfer of nucleic acids (DNA) between cyanobacterial strains and within populations (Rudi et al 1998(Rudi et al , 2000Barker et al 1999;Hayes et al 2006), combined with rapid acclimation and adaptation Hagemann et al 2001, Hagemann 2002Komárek and Kaštovský 2003) (Fig. 1).…”

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confidence: 99%

Cyanobacterial Taxonomy: Current Problems and Prospects for the Integration of Traditional and Molecular Approaches

Komárek¹

2006

ALGAE

147

115

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Cyanobacteria (Cyanophytes, Cyanoprokaryotes) are among the most fascinating organisms in the Earth's biosphere. Their origin in the Early Precambrian was one of the most important steps in evolution (Schopf 1974a(Schopf , 1974b(Schopf , 1993(Schopf , 1996. They are prokaryotic bacteria, however, their cells developed the plant-type photosynthetic apparatus that included chlorophyl a and both photosystems. The subsequent evolution of the plant kingdom is based on numerous intracellular symbioses of prokaryotic cyanobacteria with eukaryotic heterotrophs. The relative ease with which cyanobacteria enter into symbioses is a characteristic of this lineage (cf., e.g., reviews of Janson 2002; Carpenter and Foster 2002, and others). Other remarkable adaptations developed in cyanobacteria enabled them to colonize variable and extreme habitats in Earth ecosystems (Carr and Whitton 1973;Fogg et al. 1973; Whitton and Potts 2000; Rai and Gaur 2001). Cyanobacteria are the only oxyphototrophic organisms to contain Nif-genes, and mechanisms and adaptations for fixation of gaseous nitrogen. They participate in the formation of travertine and stromatolites, and a substantial part of limestone deposits over the Earth results from their metabolic activity. Several types are adapted to colonise hypersaline locations, hot springs up to over 70°C, or to enormous oscillations of temperatures and periodical drying in both cold and hot deserts. They are able to produce toxins, which can function in competitive interactions in various ecosystems. Most noteworthy is the extraordinary vitality of this special bacterial group which has not declined during its existence that has spanned billions of years.The massive developments of cyanobacterial populations have also become important because of increasing eutrophication of the biosphere. Thus there is major concern regarding "water blooms" in freshwaters of all continents, unexpected developments of cyanobacterial types in plankton and littoral zones of seas and oceans (e.g., picoplanktic Prochlorococcus, or periphytic Lyngbya majuscula), and cyanobacterial crusts in semideserts (Leptolyngbya, Microcoleus, Nostoc). From a scientific perspective the most challenging questions revolve around evolutionary strategies, the survival and diversification of cyanobacteria over the long period of their existence, and evaluation of their present diversity in nature (cf. The application of modern ecological, ultrastructural and molecular methods, aided by the cultivation of numerous cyanobacterial morphotypes, has substantially changed our knowledge of these organisms. It has led to major advances in cyanobacterial taxonomy and criteria for their phylogenetic classification. Molecular data provide basic criteria for cyanobacterial taxonomy; however, a correct phylogenetic system cannot be constructed without combining genetic data with knowledge from the previous 150 years research of cyanobacterial diversity. Thus, studies of morphological variation in nature, and modern morphological, ultrastructural...

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Biochemical Characterization of Glucosylglycerol-Phosphate Synthase of Synechocystis sp. Strain PCC 6803: Comparison of Crude, Purified, and Recombinant Enzymes

Cited by 35 publications

References 26 publications

Glucosylglycerate Biosynthesis in the Deepest Lineage of the Bacteria : Characterization of the Thermophilic Proteins GpgS and GpgP from Persephonella marina

Glucosylglycerate Biosynthesis in the Deepest Lineage of the Bacteria : Characterization of the Thermophilic Proteins GpgS and GpgP from Persephonella marina

The Bacterium Thermus thermophilus , Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate

Cyanobacterial Taxonomy: Current Problems and Prospects for the Integration of Traditional and Molecular Approaches

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