Intermolecular cleavage by UmuD-like enzymes: Identification of residues required for cleavage and substrate specificity

The UmuD-like proteins are best characterized for their role in damage-indu ced SOS mutagenesis. An essential step in this process is the enzymatic sel f-processing of the UmuD-like proteins. This reaction is thought to occur e ither via an intramolecular or intermolecular self-cleavage mechanism. Here , we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction. The Escherichia coli UmuD enzyme demonstrated the broade st substrate specificity, cleaving both E. coli and Salmonella typhimurium UmuD substrates in vivo. In comparison, the wild-type S. typhimurium UmuD ( UmuD(St)) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage. Heterologous cleavage by the UmuD(St) enzyme was, however, observed with chimeric UmuD substrates that possess r esidues 30-55 of UmuD(St). We have further localized the residue predominan tly responsible for UmuD(St)-catalyzed heterologous cleavage to Ser50 in th e substrate molecule. We hypothesize that changes at this residue affect th e positioning of the cleavage site of a substrate molecule within the catal ytic cleft of the UmuD(St) enzyme by affecting the formation of a so-called UmuD "filament-dimer". This hypothesis is further supported by the observa tion that mutations known to disrupt an E. coli UmuD' filament dimer also b lock intermolecular UmuD(Ec) cleavage. (C) 1999 Academic Press.