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Journal of Bacteriology, September 2008, p. 6153-6161, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00658-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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 ^,

Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain,¹ Departamento de Química, Universidade de Évora, Évora, Portugal,² Servicio de Microbiología, Complejo Hospitalario Universitario de Badajoz, Servicio Extremeño de Salud, Badajoz, Spain³

Received 11 May 2008/ Accepted 6 July 2008

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.

Published ahead of print on 18 July 2008.

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