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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{triangledown} ,{dagger}

Isabel Alves-Pereira,1,2 José Canales,1 Alicia Cabezas,1 Paloma Martín Cordero,3 María Jesús Costas,1 and José Carlos Cameselle1*

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 (UshAYi) of the Y. intermedia ushA gene (ushAYi) 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 (UshAEc). Therefore, UshAEc was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshAEc and UshAYi 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 kcat/Km ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshAEc conformers and the respective homology models constructed for UshAYi, 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.

* Corresponding author. Mailing address: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Apartado 108, 06080 Badajoz, Spain. Phone: 34-924289470. Fax: 34-924289468. E-mail: camselle{at}unex.es

{triangledown} Published ahead of print on 18 July 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

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.





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