Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough

Journal Article · Tue Jan 01 00:00:00 EST 1991 · Journal of Bacteriology; (USA)

OSTI ID:5476605

Pollock, W B.R.; Voordouw, G ^[1]; Loutfi, M; Bruschi, M ^[2]; Rapp-Giles, B J; Wall, J D ^[3]

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1. The Univ. of Calgary, Alberta (Canada)
2. Centre National de la Recherche Scientifique, Cedex (France)
3. Univ. of Missouri, Columbia (USA)

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc{sub 3} from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc{sub 3} gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH{sub 2}-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. Since the data indicate that cytochrome cc{sub 3} and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c{sub 3} and Hme from D. vulgaris Hildenborough suggest that the latter contains three cytochrome c{sub 3}-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene produce with covalently bound hemes.

• DOE Contract Number: FG02-87ER13713
• OSTI ID: 5476605
• Journal Information: Journal of Bacteriology; (USA), Vol. 173:1; ISSN 0021-9193
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CYTOCHROMES
GENES
DESULFOVIBRIO
MOLECULAR BIOLOGY
AMINO ACID SEQUENCE
DNA SEQUENCING
DNA-CLONING
HEME
HISTIDINE
PLASMIDS
RECOMBINANT DNA
AMINO ACIDS
AZOLES
BACTERIA
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CLONING
DNA
DNA HYBRIDIZATION
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYBRIDIZATION
IMIDAZOLES
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PIGMENTS
PORPHYRINS
PROTEINS
STRUCTURAL CHEMICAL ANALYSIS
SULFATE-REDUCING BACTERIA
550200* - Biochemistry