Back to Search
Start Over
Isolation and analysis of the gene encoding the pyruvate-ferredoxin oxidoreductase of Desulfovibrio africanus, production of the recombinant enzyme in Escherichia coli, and effect of carboxy-terminal deletions on its stability
- Source :
- Journal of bacteriology. 179(18)
- Publication Year :
- 1997
-
Abstract
- Previous studies have shown that the pyruvate-ferredoxin oxidoreductase (POR) of the sulfate-reducing bacterium Desulfovibrio africanus is a homodimer that contains one thiamine pyrophosphate and three [4Fe-4S]2+/1+ centers/subunit. Interestingly, the enzyme isolated from a strictly anaerobic bacterium is highly stable in the presence of oxygen, in contrast to the other PORs characterized in anaerobic organisms (L. Pieulle, B. Guigliarelli, M. Asso, F. Dole, A. Bernadac, and E. C. Hatchikian, Biochim. Biophys. Acta 1250:49-59, 1995). We report here the determination of the nucleotide sequence of the por gene encoding the D. africanus POR. The amino acid sequence deduced from this nucleotide sequence corresponds to the first primary structure of a homodimeric POR from strictly anaerobic bacteria. The subunit of the D. africanus POR contains two ferredoxin-type [4Fe-4S] cluster binding motifs (CX2CX2CX3CP) and four additional highly conserved cysteines belonging to a nontypical motif. These 12 cysteine residues may coordinate the three Fe-S centers present in D. africanus POR. The thiamine pyrophosphate binding domain is located in the C-terminal part of the protein close to the four conserved cysteine residues. The D. africanus enzyme sequence appears homologous to the other POR sequences. However, the enzyme differs from all other PORs by a C-terminal extension of about 60 residues of its polypeptide chain. The two cysteine residues located in this additional region may be involved in the formation of a disulfide bridge associated with the activation process of the catalytic activity. The por gene has been expressed, for the first time, in anaerobically grown Escherichia coli behind the isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, resulting in the production of POR in its active form. The recombinant enzyme is stable toward oxygen during several days, and initial characterization of the recombinant POR showed that its activity increased in the presence of dithioerythritol. These properties indicate that the recombinant POR behaves like the native D. africanus enzyme. The study of carboxy-terminal deletion mutants strongly suggests that deletions in the C-terminal region of D. africanus enzyme can have dramatic effects on the stability of the enzyme toward oxygen.
- Subjects :
- Protein Denaturation
Pyruvate Synthase
Molecular Sequence Data
medicine.disease_cause
Microbiology
Peptide Mapping
Structure-Activity Relationship
Bacterial Proteins
Oxidoreductase
medicine
Escherichia coli
Amino Acid Sequence
Molecular Biology
Peptide sequence
Sequence Deletion
chemistry.chemical_classification
Pyruvate synthase
biology
Base Sequence
Sequence Homology, Amino Acid
Nucleic acid sequence
Protein primary structure
Ketone Oxidoreductases
Recombinant Proteins
chemistry
Biochemistry
Genes, Bacterial
biology.protein
Desulfovibrio
Anaerobic bacteria
Sequence Alignment
Thiamine pyrophosphate binding
Research Article
Subjects
Details
- ISSN :
- 00219193
- Volume :
- 179
- Issue :
- 18
- Database :
- OpenAIRE
- Journal :
- Journal of bacteriology
- Accession number :
- edsair.doi.dedup.....37675a64a7cd4249f4f0ccb8aa594cce