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

Abstract

ABSTRACT 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.