Cloning, Sequence Analysis and Overexpression of the Rhodanese Gene of Azotobacter vinelandii

Abstract

A gene encoding rhodanese (rhdA) was cloned from Azotobacter vinelaizdii 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 thiosu1fate:cyanide sulfurtransferase activity increased > 600 fold in cellfree 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.

Thiosulfate sulfurtransferase-like domain–containing 1 protein interacts with thioredoxin

“…10B). The single domain rhodanese proteins GlpE, ThiI and RhdA also exhibit sulfur transfer activity with thiosulfate as a donor (42,49,50). For example, ThiI reportedly carries sulfur between IscS and tRNA.…”

Enzymatic Activity of the Arabidopsis Sulfurtransferase Resides in the C-Terminal Domain But Is Boosted by the N-Terminal Domain and the Linker Peptide in the Full- Length Enzyme

“…Only the Azotobacter ST showed unique properties among the characterized STs because it accepted almost exclusively thiosulfate with a 1×10 3 fold higher specific activity than with 3-MP (Colnaghi et al, 1996). Probably the unique stretch of amino acids around the active site is responsible for this substrate specificity Pagani et al, 2000).…”

Common themes and variations in the rhodanese superfamily

“…The extreme indetermination in attributing a defined role to STs is likely due to the fact that the identification of the in vivo substrates has thus far proven inconclusive. It appears unlikely that privileged natural substrates of rhodaneses are those identified by in vitro reactions given that the thiosulfate/mercaptopyruvate and cyanide affinity is in the millimolar range (Table 1), thus apparently incompatible with the supposed physiological role in enzymatic cyanide detoxification (8,25,39). Therefore, alternative functions, including sulfur and selenium metabolism and biosynthesis of prosthetic groups in iron-sulfur proteins, have been proposed (40 -46).…”