Cloning, sequence analysis and overexpression of the rhodanese gene of Azotobacter vinelandii. 1996

R Colnaghi, and S Pagani, and C Kennedy, and M Drummond
Dipartimento di Scienze Molecolari Agroalimentari and Centro Interuniversitario per lo Studio delle Macromolecole Informazionali, University of Milano, Italy.

A gene encoding rhodanese (rhdA) was cloned from Azotobacter vinelandii on a 2.3-kb SphI fragment. This fragment was identified by its hybridization to a PCR product obtained by amplification of genomic DNA using degenerate primers encoding the N-terminal sequence of rhodanese purified from A. vinelandii. The sequence of a 1.2-kb region revealed an 813-bp open reading frame that encoded a polypeptide of 271 amino acids, the N-terminal sequence of which was identical to that of A. vinelandii rhodanese. In a search of database entries, eukaryotic rhodaneses and rhodanese-like proteins from bacteria gave the highest scores of identity (27-30%) with the predicted product of the 813-bp open reading frame. A. vinelandii RhdA shows less sequence similarity to vertebrate rhodaneses than it does to prokaryotic rhodanese-like proteins which did not show typical rhodanese activity. Basic residues thought to be catalytically important in bovine rhodanese are not conserved in A. vinelandii rhodanese. The sequence similarity between the two structurally similar domains of rhodanese is more pronounced for the A. vinelandii enzyme than the bovine enzyme, and supports the hypothesis that the complete structure was originally generated by gene duplication. When rhdA was overexpressed in Escherichia coli, rhodanese represented 30% of total cell protein and thiosulfate:cyanide sulfurtransferase activity increased >600 fold in cell-free extracts. A. vinelandii rhdA insertion/deletion mutants had no discernible phenotype distinct from the wild-type strain with respect to growth on various sulfur sources or nitrogenase activity. Mutants retained 20% of wild-type rhodanese thiosulfate:cyanide sulfurtransferase activity suggesting the presence of redundant sulfurtransferase enzymes in A. vinelandii.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005798 Genes, Bacterial The functional hereditary units of BACTERIA. Bacterial Gene,Bacterial Genes,Gene, Bacterial
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D013045 Species Specificity The restriction of a characteristic behavior, anatomical structure or physical system, such as immune response; metabolic response, or gene or gene variant to the members of one species. It refers to that property which differentiates one species from another but it is also used for phylogenetic levels higher or lower than the species. Species Specificities,Specificities, Species,Specificity, Species
D013884 Thiosulfate Sulfurtransferase An enzyme that catalyzes the transfer of the planetary sulfur atom of thiosulfate ion to cyanide ion to form thiocyanate ion. EC 2.8.1.1. Rhodanese,Thiosulfate Cyanide Transsulphurase,Thiosulfate Sulphurtransferase,Cyanide Transsulphurase, Thiosulfate,Sulfurtransferase, Thiosulfate,Sulphurtransferase, Thiosulfate,Transsulphurase, Thiosulfate Cyanide

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