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 exampleof conserved structures with possible functional diversity,are structurally related to the catalytic subunit of Cdc25 phosphataseenzymes. The major difference characterizing the active-siteof the Azotobacter vinelandii rhodanese RhdA, with respect tothe closely related Cdc25s (A, B, C), is that in Cdc25 phosphatasesthe active site loop His–Cys–(X)₅–Arg] isone residue longer than in RhdA His–Cys–(X)₄–Arg].According to the hypothesis that the length of the RhdA active-siteloop should play a key role in substrate recognition and catalyticactivity, RhdA scaffold was the starting point for producingmutants with single-residue insertion to generate the catalyticloop HCQTHAHR (in RhdA-Ala) and HCQTHSHR (in RhdA-Ser). Analysesof the catalytic performances of the engineered RhdAs revealedthat elongation of the catalytic loop definitely compromisedthe ability to catalyze sulfur transfer reactions, while itgenerated ‘phosphatase’ enzymes able to interactproductively with the artificial substrate 3-O-methylfluoresceinphosphate. Although this study is restricted to an example ofrhodanese modules (RhdA), it provided experimental evidenceof the hypothesis that a specific mutational event (a single-residueinsertion or deletion in the active-site loop) could changethe selectivity from sulfur- to phosphate-containing substrates(or vice versa). Received February 17, 2003; revised May 30, 2003; accepted June 6, 2003.