Distinct physiological roles for the two l-asparaginase isozymes of Escherichia coli

•Escherichia coli contains two l-asparaginase isozymes with distinct localization, kinetics and regulation.•Mutant strains were used to examine the roles of these enzymes in l-asparagine utilization.•We report that l-asparaginase II permits growth on asparagine and glycerol under anaerobic conditions.•We propose that this enzyme is the first step in a co-regulated pathway leading to fumarate.•The pathway is regulated by anaerobiosis and cAMP and provides a terminal elector acceptor. Escherichia coli expresses two l-asparaginase (EC 3.5.1.1) isozymes: l-asparaginse I, which is a low affinity, cytoplasmic enzyme that is expressed constitutively, and l-asparaginase II, a high affinity periplasmic enzyme that is under complex co-transcriptional regulation by both Fnr and Crp. The distinct localisation and regulation of these enzymes suggest different roles. To define these roles, a set of isogenic mutants was constructed that lacked either or both enzymes. Evidence is provided that l-asparaginase II, in contrast to l-asparaginase I, can be used in the provision of an anaerobic electron acceptor when using a non-fermentable carbon source in the presence of excess nitrogen.