Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491.
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 degrees 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.
Fourrat
Latifa
L
Laboratoire de Physiologie et Génétique moléculaire, Département de Biologie, Faculté des Sciences Aïn-Chock, Université Hassan-II, Casablanca, Morocco.
Iddar
Abdelghani
A
Valverde
Federico
F
Serrano
Aurelio
A
Soukri
Abdelaziz
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000235:genetics*; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)