Dissection of the bifunctional Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphateacetyltransferase activity and cell growth

The bifunctional N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) e nzyme catalyzes both the acetylation of glucosamine 1-phosphate and the uri dylation of N-acetylglucosamine 1-phosphate, two subsequent steps in the pa thway for UDP-N-acetylglucosamine synthesis in bacteria. In our previous wo rk describing its initial characterization in Escherichia coli, we proposed that the 456-amino acid (50.1 kDa) protein might possess separate uridyltr ansferase (N-terminal) and acetyltransferase (C-terminal) domains, In the p resent study, we confirm this hypothesis by expression of the two independe ntly folding and functional domains. A fragment containing the N-terminal 3 31 amino acids (Tr331, 37,1 kDa) has uridyltransferase activity only, with steady-state kinetic parameters similar to the full-length protein. Further deletion of 80 amino acid residues at the C terminus results in a 250-amin o acid fragment (28.6 kDa) still exhibiting significant uridyltransferase a ctivity. Conversely, a fragment containing the 233 C-terminal amino acids ( 24.7 kDa) exhibits acetyltransferase activity exclusively. None of these in dividual domains could complement a chromosomal glmU mutation, indicating t hat each of the two activities is essential for cell viability. Analysis of truncated GlmU proteins by gel filtration further localizes regions of the protein involved in its trimeric organization. Interestingly, overproducti on of the truncated Tr331 protein in a wild-type strain results in a rapid depletion of endogenous acetyltransferase activity, an arrest of peptidogly can synthesis and cell lysis, It is shown that the acetyltransferase activi ty of the full-length protein is abolished once trapped within heterotrimer s formed in presence of the truncated protein, suggesting that this enzyme activity absolutely requires a trimeric organization and that the catalytic site involves regions of contact between adjacent monomers, Data are discu ssed in connection with the recently obtained crystal structure of the trun cated Tr831 protein.