Evidence that elongation of the catalytic loop of the Azotobacter vinelandii rhodanese changed selectivity from sulfur- to phosphate-containing substrates.
Recent investigations have shown that the rhodanese domains, ubiquitous structural modules which might represent an example of conserved structures with possible functional diversity, are structurally related to the catalytic subunit of Cdc25 phosphatase enzymes. The major difference characterizing the active-site of the Azotobacter vinelandii rhodanese RhdA, with respect to the closely related Cdc25s (A, B, C), is that in Cdc25 phosphatases the active site loop [His-Cys-(X)5-Arg] is one residue longer than in RhdA [His-Cys-(X)4-Arg]. According to the hypothesis that the length of the RhdA active-site loop should play a key role in substrate recognition and catalytic activity, RhdA scaffold was the starting point for producing mutants with single-residue insertion to generate the catalytic loop HCQTHAHR (in RhdA-Ala) and HCQTHSHR (in RhdA-Ser). Analyses of the catalytic performances of the engineered RhdAs revealed that elongation of the catalytic loop definitely compromised the ability to catalyze sulfur transfer reactions, while it generated 'phosphatase' enzymes able to interact productively with the artificial substrate 3-O-methylfluorescein phosphate. Although this study is restricted to an example of rhodanese modules (RhdA), it provided experimental evidence of the hypothesis that a specific mutational event (a single-residue insertion or deletion in the active-site loop) could change the selectivity from sulfur- to phosphate-containing substrates (or vice versa).
Forlani
F
F
Dipartimento Scienze Molecolari Agroalimentari, Università di Milano, Via Celoria 2, 20133 Milan, Italy.
Carpen
A
A
Pagani
S
S
D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D005452:Fluoresceins / Q000378:metabolism*; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D013884:Thiosulfate Sulfurtransferase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D013885:Thiosulfates / Q000378:metabolism*
C032079:3-O-methylfluorescein phosphate; D005452:Fluoresceins; D019163:Reducing Agents; D013885:Thiosulfates; D013884:Thiosulfate Sulfurtransferase; D010744:Phosphoric Monoester Hydrolases