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Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491

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Abstract

Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram-negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of the N. meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter. The IPTG-induced GAPN was purified ca. 48-fold from E. coli cells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibiting absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic polypeptide with an isoelectric point of 6.3. The optimum pH of the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64 °C. Molecular phylogenetic data suggest that N. meningitidis GAPN has a closer relationship with archaeal GAPNs and glyceraldehyde dehydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukaryotes.

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Acknowledgments

The authors thank Prof. M. Losada for encouragement and help. This work is part of several collaborative Research Projects between the CNR (Morocco) and CSIC (Spain). It was supported by AECI (Spain) and collaborative grants of the Andalusian Government—Ministère d’Education et de la Recherche Scientifique of Morocco (Junta de Andalucía, Proyectos de Cooperación al Desarrollo en el Ámbito Universitario de la Agencia Andaluza de Cooperación Internacional A52/02, and A54/04), group PAI CVI-261 and grants from CNRST (Morocco).

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Authors and Affiliations

  1. Laboratoire de Physiologie et Génétique moléculaire, Département de Biologie, Faculté des Sciences Aïn-Chock, Université Hassan-II, Casablanca, Morocco

    Latifa Fourrat & Abdelaziz Soukri

  2. Unité Radio-Immuno-Analyse, Département des Sciences du vivant, Centre National de l’Energie, des Sciences et des Techniques Nucléaires (CNESTEN), Centre d’Etudes Nucléaires de la Maâmora, Rabat, Morocco

    Abdelghani Iddar

  3. Instituto de Bioquímica Vegetal y Fotosíntesis (CSIC-Universidad de Sevilla), Centro de Investigaciones Científicas Isla de la Cartuja, Sevilla, Spain

    Federico Valverde & Aurelio Serrano

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  1. Latifa Fourrat
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  2. Abdelghani Iddar
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  3. Federico Valverde
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  5. Abdelaziz Soukri
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Correspondence to Abdelaziz Soukri.

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Fourrat, L., Iddar, A., Valverde, F. et al. Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491. Mol Cell Biochem 305, 209–219 (2007). https://doi.org/10.1007/s11010-007-9545-z

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  • DOI: https://doi.org/10.1007/s11010-007-9545-z

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