Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803
Détails
ID Serval
serval:BIB_7AF2E4CE6FB9
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803
Périodique
Plant Molecular Biology
ISSN
0167-4412 (Print)
Statut éditorial
Publié
Date de publication
05/1998
Volume
37
Numéro
2
Pages
205-15
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: May
Research Support, Non-U.S. Gov't --- Old month value: May
Résumé
A 3.3 kb HindIII restriction-digest DNA fragment was isolated from a Synechocystis sp. strain PCC6803 subgenomic plasmid library which strongly hybridized to a 349 bp fragment of the icfA (ccaA) gene from Synechococcus sp. strain PCC7942. DNA sequence analysis of the fragment revealed three open reading frames (ORFs), two of which potentially coded for pantothenate synthetase (ORF275) and cytidylate kinase (ORF230). The third, ORF274, was 825 bp in length, encoding a deduced polypeptide of 274 aa (Mr, 30747) that bears 55% sequence identity to the Synechococcus icfA (ccaA) translation product, a beta-type carbonic anhydrase (CA). A 932 bp EcoRI fragment containing ORF274 was subcloned into an expression vector and the construct was transformed into Escherichia coli for overexpression. Electrometric assays for CA activity revealed that whole cell extracts containing the recombinant protein significantly enhanced the rate of conversion of CO2 to HCO3- and that 98% of this catalytic activity was inhibited by ethoxyzolamide, a well-characterized CA inhibitor. Antisera derived against the overexpressed protein recognized a 30.7 kDa protein that was predominantly associated with the isolated carboxysome fraction from Synechocystis. These results provide molecular and physiological evidence for the identification of a ccaA homologue in Synechocystis PCC6803 that encodes a carboxysomal beta-type CA.
Mots-clé
Amino Acid Sequence
Base Sequence
Carbonic Anhydrase Inhibitors/pharmacology
Carbonic Anhydrases/*genetics/metabolism
Cloning, Molecular
Cyanobacteria/enzymology/*genetics
DNA, Bacterial/analysis
Escherichia coli/genetics
Ethoxzolamide/pharmacology
Gene Expression
Genes, Bacterial/*genetics
Molecular Sequence Data
Open Reading Frames/genetics
Recombinant Fusion Proteins/isolation & purification/metabolism
Restriction Mapping
Sequence Analysis, DNA
Sequence Homology, Amino Acid
Sequence Homology, Nucleic Acid
Pubmed
Web of science
Création de la notice
25/01/2008 9:38
Dernière modification de la notice
20/08/2019 15:36