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

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 CDPalcohol 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.

An essential role for UshA in processing of extracellular flavin electron shuttles by Shewanella oneidensis

“…The structure of FAD, consisting of an ADP nucleotide with 5′‐linkage to a riboflavin moiety (Fig. 1), is reminiscent of UDP‐sugars and CDP‐alcohols that have been reported to be substrates of E. coli UshA (Glaser et al ., 1967; Neu, 1967; Alves‐Pereira et al ., 2008). However, despite this similarity in substrate structure, E. coli UshA exhibits very low FAD hydrolysis activity (Alves‐Pereira et al ., 2008).…”

“…1), is reminiscent of UDP‐sugars and CDP‐alcohols that have been reported to be substrates of E. coli UshA (Glaser et al ., 1967; Neu, 1967; Alves‐Pereira et al ., 2008). However, despite this similarity in substrate structure, E. coli UshA exhibits very low FAD hydrolysis activity (Alves‐Pereira et al ., 2008). In order to test whether S. oneidensis UshA has the ability to catalyse hydrolysis of FAD, we added sonicated extracts of wild‐type and Δ ushA cells to solutions of FAD.…”

“…Escherichia coli UshA has been found to hydrolyse FAD with only approximately 1% of the activity of AMP hydrolysis (Alves‐Pereira et al ., 2008). We verified that E. coli UshA hydrolyses FAD slowly using our fluorescence‐based hydrolysis assay by incubating cell extracts from wild‐type and Δ ushA E. coli strains with FAD.…”

“…We have shown, both by monitoring fluorescence increases and by HPLC analyses, that S. oneidensis UshA hydrolyses FAD into the flavin derivative FMN. FAD has not previously been reported to be a major substrate for UshA (Alves‐Pereira et al ., 2008), and we have verified that FAD is not as rapidly hydrolysed by the E. coli homologue. The difference in substrate specificities suggests that Shewanella has adapted to high periplasmic concentrations of FAD.…”

Analysis of Soluble Protein Complexes in Shigella flexneri Reveals the Influence of Temperature on the Amount of Lipopolysaccharide

“…UshA was more recently found to have a novel, highly efficient CDP-alcohol hydrolase activity (20), and it may have a general role in utilization of external UDP-glucose and extracellular nucleotides (21). X-ray structures have shown that UshA is a monomer (22); however, in our analyses it appeared as a complex with a molecular mass of ϳ177 kDa.…”