Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase.
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 gt11 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.
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Department of Biochemistry and Molecular Biology, USF College of Medicine, Tampa, FL 33612.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002708:Chlorella / Q000201:enzymology*; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006418:Heme / Q000378:metabolism*; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D012333:RNA, Messenger; D006418:Heme; D004247:DNA; D009565:Nitrate Reductases; D050901:Nitrate Reductase