Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282 000 by mutating an active site lid

Matthew R. Burrell, Victoria J. Just, Laura Bowater, Shirley A. Fairhurst, Laura Requena, David M Lawson, Stephen Bornemann

Research output: Contribution to journalArticle

44 Citations (Scopus)


Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features:  a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282 000 (SEN161-3DAS), 275 000 (SENS161-4DSSN), and 225 000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25 600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.
Original languageEnglish
Pages (from-to)12327-12336
Number of pages10
Issue number43
Publication statusPublished - 2007

Cite this