Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase

• Published in: Biochemical journal 1991-08, Vol.278 (1), p.203-209
• Main Authors: Cannons, A.C, Iida, N, Solomonson, L.P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Blotting, Northern; cDNA; Chlorella - enzymology; Chlorella vulgaris; clones; ddbj/x56771; DNA; DNA - genetics; embl/x56771; enzymatic activity; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Freshwater; Fundamental and applied biological sciences. Psychology; genbank/x56771; Gene Expression; genetics; heme; Heme - metabolism; Molecular Sequence Data; molecular structure; Nitrate Reductase; Nitrate Reductases - genetics; Nitrate Reductases - metabolism; nucleotide sequences; Oxidoreductases; Polymerase Chain Reaction; RNA, Messenger - analysis; RNA, Messenger - genetics; sequence homology; Sequence Homology, Nucleic Acid; spectral analysis; Transformation, Bacterial
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2780203

A partial cDNA clone coding for the haem-binding domain of NADH:nitrate reductase (EC 1.6.6.1) (NR) from the unicellular green alga Chlorella vulgaris has been isolated, sequenced and expressed. A 1.2 kb cDNA (pCVNR1) was isolated from a lambda gt 11 expression library produced from polyadenylated RNA extracted from nitrate-grown Chlorella cells. pCVNR1 hybridized to a 3.5 kb mRNA transcript that was nitrate-inducible and absent from ammonium-grown cells. The entire sequence of pCVNR1 was obtained and found to have a single uninterrupted reading frame. The derived amino acid sequence of 318 amino acids has a 45-50 % similarity to higher-plant NRs, including Arabidopsis thaliana, spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum). A comparison with the putative domain structure of higher-plant nitrate reductases suggested that this sequence contains the complete haem-binding domain, approximately one-third of the Mo-pterin domain and no FAD-binding domain. A 32% sequence similarity is evident when comparing the Chlorella NR haem domain with that of calf cytochrome b5. Expression of pCVNR1 in a pET vector synthesized a 35 kDa protein that was antigenic to anti-(Chlorella NR) antibody. The spectral properties of this protein (reduced and oxidized) in the 400-600 nm region are identical with those of native Chlorella NR and indicate that haem is associated with the protein.