Nucleotide sequence of the gene encoding the 33 kDa water oxidizing polypeptide in Anabaena sp. strain PCC 7120 and its expression in Escherichia coli

Abstract

The 33 kDa extrinsic polypeptide of photosystem II, also known as the manganese-stabilizing polypeptide (MSP), is located on the lumen side of the thylakoid and is involved in water oxidation. The gene for MSP, designated woxA, has been cloned from the nitrogen-fixing filamentous cyanobacterium Anabaena and sequenced. The woxA open reading frame was found to be 819 bp. The deduced amino acid sequence was 63% and 59% homologous with that of Synechococcus and Synechocystis, respectively, and 44% conserved when compared to the MSP of spinach or pea. Two cysteine residues at positions 48 and 73 were found to be conserved in cyanobacteria and plants. The first 29 amino acids are hydrophobic and may represent the transit peptide. woxA: :phoA translational fusion products, in which the body of Escherichia coli alkaline phosphatase was fused to the amino terminal portion of woxA between residues 35 and 130, yielded active alkaline phosphatase in E. coli. Thus the transit peptide of woxA functions in E. coli to transport phosphatase across the cytoplasmic membrane. S1 mapping and primer extension experiments showed that the woxA transcription initiation site is located 220 bp upstream from the translational start. The woxA promoter has some resemblance to the E. coli consensus and other known Anabaena vegetative cell promoters.