CDP-alcohol hidrolasa: Una nueva y elevada actividad de la 5´-nucleotidasa / UDP-azúcar hidrolasa codificada por el gen ushA de Yersinia intermedia y Escherichia coli

Abstract

This work is part of an effort devoted to the identification and characterization of nucleoside-diphosphate-X (NDP-X) hydrolases in a diversity of biological sources. These phosphohydrolases drive much attention by their variety, by the relationship between their catalytic specificity and structure, and by the role they may play in the disposal or in the modulation of the effects of their substrates. Very few of the known NDP-X hydrolases show preference for CDP-X. The initial aim of this work was the molecular identification and characterization of the enzyme responsible for a Mg2+-activated CDP-alcohol hydrolase activity earlier observed in Yersinia intermedia suspensions. The CDP-alcohol hydrolase was detected in the extracellular medium of Y. intermedia bacterial suspensions incubated in a buffered medium supplemented with Triton X-100. The amount of enzyme in this medium was dependent on the temperature at which cells were grown, with a maximum at 25ºC. Its purification, molecular cloning, expression and characterization proved that it is a novel and elevated activity of the 5´-nucleotidase / UDP-sugar hydrolase encoded by the ortholog of the ushA gene in Yersinia intermedia (Yi-UshA). This enzyme has been studied in detail by others in Escherichia coli (Ec-UshA) and it is known to display 5´-nucleotidase, UDP-sugar hydrolase and dinucleoside-polyphosphate hydrolase activities, but its CDP-alcohol hydrolase activity was unknown. Therefore, in addition to Yi-UshA studies, Ec-UshA was overexpressed from a plasmid containing the cloned ushA gene, and it was found it was also active as CDP-alcohol hydrolase. Far from being a minor activity, the hydrolysis of CDP-alcohols occurred with a high catalytic efficiency, particularly so with Ec-UshA (kcat/Km ≈ 108 M-1 s-1), only slightly lower than the 5´-nucleotidase activity and somewhat higher than the UDP-sugar hydrolase (after which the gene and the protein are named) and the dinucleoside polyphoshate hydrolase activities. Such high catalytic efficiencies indicate that the reaction is not limited by the chemical steps of the mechanism, but by the enzyme and substrate diffusion steps, as corresponds to so-called ‘perfect’ enzymes. In addition to what is summarized above, which makes the main line of this work, other experimental and bioinformatic results are also reported and discussed. They refer to the enzymatic behavior of Yi-UshA and Ec-UshA, to the sequence and structure of Yi-UshA and to the possible biological role of these enzymes, with regards to Y. intermedia, E. coli and other bacterial species.