Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

被引:64
作者
So, AKC [1 ]
Espie, GS [1 ]
机构
[1] Univ Toronto, Dept Bot, Mississauga, ON L5L 1C6, Canada
基金
加拿大自然科学与工程研究理事会;
关键词
carbonic anhydrase; carboxysome; cyanobacteria; cytidylate kinase; pantothenate synthetase; Synechocystis PCC6803;
D O I
10.1023/A:1005959200390
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. attain 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 (M-r, 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.
引用
收藏
页码:205 / 215
页数:11
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