Catalytic properties, stability and the structure of the conformational lock in the alkaline phosphatase from Escherichia coli

Poltorak, O. M.; Chukhray, E. S.; Torshin, I. Yu.; Atyaksheva, L. F.; Trevan, M. D.; Chaplin, Martin F.

doi:10.1016/s1381-1177(99)00039-9

Cited by 29 publications

(22 citation statements)

References 3 publications

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“…However, CmAP retained its stability and activity after pre-incubation with EDTA in concentration of 50-100 mM in the presence of Mg 2+ (data not shown). It has been suggested that CmAP does not require an excess of Mg 2+ for binding in the M3 site, which has been generally noticed in the structural reorganization that occurs upon metal binding during enzyme dimerization and activation (Janeway et al 1993;Poltorak et al 1999;Babor et al 2005). The high working efficacy and stability of a monomeric CmAP can be attributed to the intrinsically tight binding of both catalytic and structural metal ions.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

et al. 2014

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The psychrophilic marine bacterium, Cobetia marina, recovered from the mantle tissue of the marine mussel, Crenomytilus grayanus, which contained a gene encoding alkaline phosphatase (AP) with apparent biotechnology advantages. The enzyme was found to be more efficient than its counterparts and showed k cat value 10-to 100-fold higher than those of all known commercial APs. The enzyme did not require the presence of exogenous divalent cations and dimeric state of its molecule for activity. The recombinant enzyme (CmAP) production and purification were optimized with a final recovery of 2 mg of the homogenous protein from 1 L of the transgenic Escherichia coli Rosetta(DE3)/Pho40 cells culture. CmAP displayed a half-life of 16 min at 45°C and 27 min at 40°C in the presence of 2 mM EDTA, thus suggesting its relative thermostability in comparison with the known cold-adapted analogues. A high concentration of EDTA in the incubation mixture did not appreciably inhibit CmAP. The enzyme was stable in a wide range of pH (6.0-11.0). CmAP exhibited its highest activity at the reaction temperature of 40-50°C and pH 9.5-10.3. The structural features of CmAP could be the reason for the increase in its stability and catalytic turnover. We have modeled the CmAP 3D structure on the base of the high-quality experimental structure of the close homologue Vibrio sp. AP (VAP) and mutated essential residues predicted to break Mg 2+ bonds in CmAP. It seems probable that the intrinsically tight binding of catalytic and structural metal ions together with the flexibility of intermolecular and intramolecular links in CmAP could be attributed to the adapted mutualistic lifestyle in oceanic waters.

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Section: Discussionmentioning

confidence: 99%

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

et al. 2014

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“…Some oligomeric enzymes may contain a specific structure, called "conformational lock," which is defined as a complexity of inter-subunit contacts that, by progressive and stepwise breaks, leads to the separation of the inactive monomers. This structure gives additional structural/functional stability to oligomeric enzymes, due to the interaction of subunits and the formation of several active oligomeric forms [27]. In the case of OCT only trimeric form has catalytic activity, underline the importance of subunit interaction and arrangement for the formation of active catalytic site.…”

Section: Discussionmentioning

confidence: 99%

Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT

Bellocco

Barreca

Laganà

et al. 2008

International Journal of Biological Macromolecules

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“…Different subunits in oligomeric enzymes are linked to each other by special sites namely "conformational lock" [37]. Based on Poltorak theory [37][38][39], the thermal stress causes sequential disruption of conformational locks leading to subunit dissociation:…”

Section: Discussionmentioning

confidence: 99%

New Model for Polymerization of Oligomeric Alcohol Dehydrogenases into Nanoaggregates

Barzegar

Moosavi-Movahedi

Kyani

et al. 2009

Appl Biochem Biotechnol

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Polymerization and self-assembly of proteins into nanoaggregates of different sizes and morphologies (nanoensembles or nanofilaments) is a phenomenon that involved problems in various neurodegenerative diseases (medicine) and enzyme instability/inactivity (biotechnology). Thermal polymerization of horse liver alcohol dehydrogenase (dimeric) and yeast alcohol dehydrogenase (tetrameric), as biotechnological ADH representative enzymes, was evaluated for the development of a rational strategy to control aggregation. Constructed ADH nuclei, which grew to larger amorphous nanoaggregates, were prevented via high repulsion strain of the net charge values. Good correlation between the variation in scattering and lambda(-2) was related to the amorphousness of the nanoaggregated ADHs, shown by electron microscopic images. Scattering corrections revealed that ADH polymerization was related to the quaternary structural changes, including delocalization of subunits without unfolding, i.e. lacking the 3D conformational and/or secondary-ordered structural changes. The results demonstrated that electrostatic repulsion was not only responsible for disaggregation but also caused a delay in the onset of aggregation temperature, decreasing maximum values of aggregation and amounts of precipitation. Together, our results demonstrate and propose a new model of self-assembly for ADH enzymes based on the construction of nuclei, which grow to formless nanoaggregates with minimal changes in the tertiary and secondary conformations.

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Catalytic properties, stability and the structure of the conformational lock in the alkaline phosphatase from Escherichia coli

Cited by 29 publications

References 3 publications

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT

New Model for Polymerization of Oligomeric Alcohol Dehydrogenases into Nanoaggregates

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