Characterization of a rhodanese from the cyanogenic bacterium Pseudomonas aeruginosa.
Pseudomonas aeruginosa, the rRNA group I type species of genus Pseudomonas, is a Gram-negative, aerobic bacterium responsible for serious infection in humans. P. aeruginosa pathogenicity has been associated with the production of several virulence factors, including cyanide. Here, the biochemical characterization of recombinant P. aeruginosa rhodanese (Pa RhdA), catalyzing the sulfur transfer from thiosulfate to a thiophilic acceptor, e.g., cyanide, is reported. Sequence homology analysis of Pa RhdA predicts the sulfur-transfer reaction to occur through persulfuration of the conserved catalytic Cys230 residue. Accordingly, the titration of active Pa RhdA with cyanide indicates the presence of one extra sulfur bound to the Cys230 Sgamma atom per active enzyme molecule. Values of K(m) for thiosulfate binding to Pa RhdA are 1.0 and 7.4mM at pH 7.3 and 8.6, respectively, and 25 degrees C. However, the value of K(m) for cyanide binding to Pa RhdA (=14 mM, at 25 degrees C) and the value of V(max) (=750 micromol min(-1)mg(-1), at 25 degrees C) for the Pa RhdA-catalyzed sulfur-transfer reaction are essentially pH- and substrate-independent. Therefore, the thiosulfate-dependent Pa RhdA persulfuration is favored at pH 7.3 (i.e., the cytosolic pH of the bacterial cell) rather than pH 8.6 (i.e., the standard pH for rhodanese activity assay). Within this pH range, conformational change(s) occur at the Pa RhdA active site during the catalytic cycle. As a whole, rhodanese may participate in multiple detoxification mechanisms protecting P. aeruginosa from endogenous and environmental cyanide.
Cipollone
Rita
R
Dipartimento di Biologia, Università Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy.
Bigotti
Maria Giulia
MG
Frangipani
Emanuela
E
Ascenzi
Paolo
P
Visca
Paolo
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013455:Sulfur / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013455:Sulfur; D013884:Thiosulfate Sulfurtransferase