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-phosphate Acetyltransferase Activity and Cell Growth

Abstract

The bifunctional N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) enzyme catalyzes both the acetylation of glucosamine 1-phosphate and the uridylation of N-acetylglucosamine 1-phosphate, two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis in bacteria. In our previous work describing its initial characterization in Escherichia coli, we proposed that the 456-amino acid (50.1 kDa) protein might possess separate uridyltransferase (N-terminal) and acetyltransferase (C-terminal) domains. In the present study, we confirm this hypothesis by expression of the two independently folding and functional domains. A fragment containing the N-terminal 331 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-amino acid fragment (28.6 kDa) still exhibiting significant uridyltransferase activity. Conversely, a fragment containing the 233 C-terminal amino acids (24.7 kDa) exhibits acetyltransferase activity exclusively. None of these individual domains could complement a chromosomal glmU mutation, indicating that 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, overproduction of the truncated Tr331 protein in a wild-type strain results in a rapid depletion of endogenous acetyltransferase activity, an arrest of peptidoglycan synthesis and cell lysis. It is shown that the acetyltransferase activity of the full-length protein is abolished once trapped within heterotrimers 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 discussed in connection with the recently obtained crystal structure of the truncated Tr331 protein.

Protein signatures distinctive of chlamydial species: horizontal transfers of cell wall biosynthesis genes glmU from archaea to chlamydiae and murA between chlamydiae and Streptomyces a aThe GenBank accession numbers for the sequences reported in this paper are indicated in the text.

“…In this context, the presence of large inserts in essential enzymes involved in cell wall synthesis has the potential to inactivate or modify the cellular functions of such proteins. It should be pointed out in this regard, that GlmU is a bifunctional protein which catalyses two of the essential steps leading to the synthesis of UDP-N-acetylglucosamine, a fundamental precursor for bacterial cell wall synthesis (Gehring et al, 1996 ;Pompeo et al, 2001). It has been shown that the N-terminal domain of this protein (residues 3-227 in E. coli) catalyses the uridyltransferase activity whereas the C-terminal domain is responsible for the acetyltransferase activity (Brown et al, 1999 ;Pompeo et al, 2001).…”

“…In the protein UDP-N-acetylglucosamine pyrophosphorylase (GlmU), which catalyses both the acetylation of glucosamine 1-phosphate and the uridylation of N-acetylglucosamine 1-phosphate to produce UDP-N-acetylglucosamine (Gehring et al, 1996 ;Pompeo et al, 2001), a 17 aa indel in a conserved region has been identified that is commonly shared by various archaea and chlamydial species (Fig. 3).…”

Regulation of Protein-Carbohydrate Interactions in Bacteria and Fungi

“…It was demonstrated that the acetyltransferase activity of the full-length protein is suppressed when embedded within heterotrimers produced in the presence of the truncated protein; thus, suggesting that the enzymatic activity necessitates a trimeric organization, with the catalytic site involved in contact sites between adjacent monomers. It was demonstrated that trimerization was perspicuously necessary for glucosamine-1-phosphateacetyltransferase activity and cell growth [14].…”

“…N-acetylglucosamine-1-phosphate uridyltransferase (Gimu) exhibits the properties of a bifunctional enzyme that catalyses both the acetylation of glucosamine 1-phosphate and uridylation of N-acetylglucosamine 1-phosphate, as well as two concomitant levels in the UDP-N-acetylglucosamine synthesis pathway in bacteria [14]. It is proposed that the 456-amino acid (50.1kDa) protein may contain disparate uridyltransferase (N-terminal) and acetyltransferase (C-terminal) domains; which is confirmed via expression of the two distinct folding and functional domains.…”

Self‐association studies of the bifunctional N‐acetylglucosamine‐1‐phosphate uridyltransferase from Escherichia coli

“…The two enzymatic activities are contained within two distinct domains of the GlmU protein and are independent of each other. 3,4 The acetyl transferase activity is carried out by the C-terminal domain, which adopts a trimeric left-handed parallel beta helix (LbH) fold. 5,6 The uridyl transferase activity is contained within the globular N-terminal domain, which is part of the nucleotide triphosphate transferase superfamily of enzymes that resemble the Rossman fold and require magnesium for catalysis.…”