Characterization of substrate binding and catalysis in the potential antibacterial target N‐acetylglucosamine‐1‐phosphate uridyltransferase (GlmU)

Abstract

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the first step in peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. The products of the GlmU reaction are essential for bacterial survival, making this enzyme an attractive target for antibiotic drug discovery. A series of Haemophilus influenzae GlmU (hiGlmU) structures were determined by X-ray crystallography in order to provide structural and functional insights into GlmU activity and inhibition. The information derived from these structures was combined with biochemical characterization of the K25A, Q76A, D105A, Y103A, V223A, and E224A hiGlmU mutants in order to map these active-site residues to catalytic activity of the enzyme and refine the mechanistic model of the GlmU uridyltransferase reaction. These studies suggest that GlmU activity follows a sequential substratebinding order that begins with UTP binding noncovalently to the GlmU enzyme. The uridyltransferase active site then remains in an open apo-like conformation until N-acetylglucosamine-1-phosphate (GlcNAc-1-P) binds and induces a conformational change at the GlcNAc-binding subsite. Following the binding of GlcNAc-1-P to the UTP-charged uridyltransferase active site, the non-esterified oxygen of GlcNAc-1-P performs a nucleophilic attack on the a-phosphate group of UTP. The new data strongly suggest that the mechanism of phosphotransfer in the uridyltransferase reaction in GlmU is primarily through an associative mechanism with a pentavalent phosphate intermediate and an inversion of stereochemistry. Finally, the structural and biochemical characterization of the uridyltransferase active site and catalytic mechanism described herein provides a basis for the structure-guided design of novel antibacterial agents targeting GlmU activity.

The Three-dimensional Structure of a Mycobacterial DapD Provides Insights into DapD Diversity and Reveals Unexpected Particulars about the Enzymatic Mechanism

“…The overall dimensions of the Mtb-DapD trimer are about 65 Å × 72 Å × 75 Å, and the total surface area buried upon trimer formation is about 10,000 Å 2 . This means that about 25% of the total surface of each subunit is engaged in the formation slightly distorted due to a bulge formation (at residues [26][27][28][29] and β3 harbors an insertion, which includes a short α-helix (α1; residues [49][50][51][52]. This large β-sheet wraps around helix α2 (residues 82-94).…”

NMR solution structure of poliovirus uridylyated peptide linked to the genome (VPgpU)

“…A crystal structure for a different enzyme, N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) in complex with UDP-N-acetylglucosamine, shows that the side chain of a conserved Gln residue (that is essential for activity) forms hydrogen bonds to the uridine base[38]. Similarly, our VPgpU structure shows the side chain of Gln22 positioned on the “back side” of the uridine base, i.e.…”

Structures ofBacteroides fragilisuridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA)

“…While no previous LpxA structures contained a divalent cation at the threefold axis, the presence of a divalent ion situated on the threefold axis of other left-handed -helical folds is observed in N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) structures (Mochalkin et al, 2007(Mochalkin et al, , 2008Sulzenbacher et al, 2001;Olsen et al, 2007;Doig et al, 2014;Olsen & Roderick, 2001;Jagtap et al, 2013;Kostrewa et al, 2001) and that of -class carbonic anhydrase (Jeyakanthan et al, 2008).…”