Deciphering the Function of Enzymes Related to Salmonella Infection in the Gut of Cattle

Abstract

Non-typhoidal Salmonella can colonize gastrointestinal system of cattle and can also cause significant food-borne disease in humans. The use of a library of single-gene deletions (SGD) in Salmonella enterica serotype Typhimurium allowed identification of several proteins that are under selection in the intestine of cattle. STM2437 (yfeJ) encodes one of these proteins and it is currently annotated as a Type I glutamine amidotransferase. STM2437 was purified to homogeneity and its catalytic properties with a wide range of γ-glutamyl derivatives were determined. Catalytic efficiency towards the hydrolysis of L-glutamine was extremely weak with a value of kcat/Km of 20 M⁻¹ s⁻¹. γ-L-Glutamyl hydroxamate was identified as the best substrate for STM2437, with a value of kcat/Km of 9.6 x 10⁴ M⁻¹ s⁻¹. A homology model of STM2437 was constructed based on the known crystal structure of a protein of unknown function (PDB id: 3L7N) and γ-L-glutamyl hydroxamate was docked into the active site. Acivicin was shown to inactivate the enzyme by reaction with the active site cysteine residue and the subsequent loss of HCl. Mutation of Cys91 to serine completely abolished catalytic activity. Inactivation of STM2437 did not affect the ability of this strain to colonize mice, but it inhibited the growth of S. typhimurium in bacteriologic media containing. Thus, although STM2437 does not appear to play a role in the murine models we tested, it is necessary for growth in the presence of γ-L-glutamyl hydroxamate. Of other genes identified, a cluster of genes is related to carbohydrate metabolism and transportation. It is proposed that the incoming carbohydrate is first phosphorylated by a hypothetical phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). The metabolite is further phosphorylated by the kinase STM3781, and then cleaved by the aldolase STM3780. The aldolase was purified to homogeneity, and its aldol condensation activity with a wide range of aldehydes was determined. Surprisingly, STM3780 can condense two molecules of dihydroxyacetone phosphate (DHAP) into dendroketose bisphosphate. The retro-aldol catalytic efficiency towards fructose bisphosphate was extremely weak with a value of kcat/Km of 28 M⁻¹ s⁻¹. The complete characterization of this gene cluster remains elusive.