Swit-4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1

The Gram-negative bacterium Sphingomonas wittichii RW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, the S. wittichii genome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit-4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit-4259 is a small (28.8?kDa) tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. Structural characterization shows that the tertiary structure of Swit-4259 is nearly identical to that of the true decarboxylases, but there are important differences in the fine structure of the Swit-4259 active site that lead to a divergence in function. In addition, it is shown that while it is a poor substrate, Swit-4259 can catalyze the hydration of 2-oxo-hex-3-enedioate to yield 2-oxo-4-hydroxyhexanedioate. It is also demonstrated that Swit-4259 has pyruvate aldolase-dehydratase activity, a feature that is common to all of the family V ADCSF enzymes studied to date. The enzymatic activity, together with the genomic context, suggests that Swit-4259 may be a hydratase with a role in the metabolism of an as-yet-unknown hydrocarbon. These data have implications for engineering bioremediation pathways to degrade specific pollutants, as well as structure-function relationships within the ADCSF in general.The acetoacetate decarboxylase-like superfamily contains a subset of proteins that are predicted to be involved in secondary metabolism based on gene context. Swit-4259, a protein of unknown function, belongs to this emerging family V subset, in which the overall fold resembles prototypical acetoacetate decarboxylases, but the active-site architecture leads to a divergence in function.