CDP-alcohol hydrolase, a very efficient activity of the 5'-nucleotidase/UDP-sugar hydrolase encoded by the ushA gene of Yersinia intermedia and Escherichia coli

Nucleoside 5′-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25°C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5′-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA_Yi) of the Y. intermedia ushA gene (ushA_Yi) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshA_Ec). Therefore, UshA_Ec was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA_Ec and UshA_Yi behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with k_cat/K_m ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA_Ec conformers and the respective homology models constructed for UshA_Yi, and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.