Substitution of aspartic acid-217 of Citrobacter freundii cephalosporinase and properties of the mutant enzymes.

On the assumption that Asp-217 of a Citrobacter freundii cephalosporinase forms a salt-bridge with the conserved Lys-67, Asp-217 was changed to glutamic acid, threonine or lysine. The mutant enzymes retained about the same level of activity as that of the wild-type enzyme, and the participation of Asp-217 in the salt-bridge was ruled out. However, the mutations resulted in an increase in hydrolytic activity toward oxyimino-cephalosporins such as cefuroxime, cefmenoxime and ceftazidime, suggesting a possible mechanism of the bacterial resistance to the novel beta-lactams by a single mutation in cephalosporinases.