GlmU (N-acetylglucosamine-1-phosphate uridyltransferase) bound to three magnesium ions and ATP at the active site

Abstract

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme exclusive to prokaryotes, belongs to the family of sugar nucleotidyltransferases (SNTs). The enzyme binds GlcNAc-1-P and UTP, and catalyzes a uridyltransfer reaction to synthesize UDP-GlcNAc, an important precursor for cell-wall biosynthesis. As many SNTs are known to utilize a broad range of substrates, substrate specificity in GlmU was probed using biochemical and structural studies. The enzymatic assays reported here demonstrate that GlmU is specific for its natural substrates UTP and GlcNAc-1-P. The crystal structure of GlmU bound to ATP and GlcNAc-1-P provides molecular details for the inability of the enzyme to utilize ATP for the nucleotidyltransfer reaction. ATP binding results in an inactive pre-catalytic enzyme-substrate complex, where it adopts an unusual conformation such that the reaction cannot be catalyzed; here, ATP is shown to be bound together with three Mg2+ ions. Overall, this structure represents the binding of an inhibitory molecule at the active site and can potentially be used to develop new inhibitors of the enzyme. Further, similar to DNA/RNA polymerases, GlmU was recently recognized to utilize two metal ions, MgA2+ and MgB2+, to catalyze the uridyltransfer reaction. Interestingly, displacement of MgB2+ from its usual catalytically competent position, as noted in the crystal structure of RNA polymerase in an inactive state, was considered to be a key factor inhibiting the reaction. Surprisingly, in the current structure of GlmU MgB2+ is similarly displaced; this raises the possibility that an analogous inhibitory mechanism may be operative in GlmU.

Keywords: ATP binding; enzyme inhibitor; substrate specificity; sugar nucleotidyltransferase.

Figure 1

A nucleotidyltransfer assay for GlmU^Mtb was carried out for different nucleotides and sugar phosphates. (a) The ability of GlmU^Mtb to utilize different nucleotides (2 mM) is shown. The maximum enzymatic activity was observed for UTP, which is the natural substrate; considering this as 100%, the activities of the other nucleotides are shown as relative percentage activity. Modified malachite green assays were carried out in triplicate and error bars are shown. (b) Similarly, to test for its ability to use different sugars, assays were carried out with UTP as the substrate. Uridyltransfer assays were carried out with three sugar phosphates (indicated) at 2 mM concentration. The maximum activity was observed with the natural substrate GlcNAc-1-P; considering this as 100%, the products formed when glucose-1-P and mannose-1-P were used are shown.

Figure 2

(a) The structure of GlmU^Mtb[GlcNAc-1-P:ATP] is shown. The acetyltransferase and uridyltransferase active sites are marked A and U. The inset shows an enlarged view of the uridyltransfer active site, with unbiased electron density for the ligands in an F _o − F _c map contoured at 2σ. The map was generated using F _o − F _c coefficients and phases calculated before including the ligands and Mg²⁺ ions (but after initial refinement of the protein atoms alone). Modelled into the density are ATP, GlcNAc-1-P and the three Mg²⁺ ions, which are labelled Mg_A ²⁺, Mg_B′ ²⁺ and Mg_C ²⁺. (b) A structural comparison of GlmU^Mtb[GlcNAc-1-P:ATP] with GlmU^Mtb[GlcNAc-1-P:UTP] is shown as ribbons coloured purple and pink, respectively. The nucleotides ATP and UTP bound at the active site are shown as sticks coloured blue and magenta, respectively. The loop regions that show a conformational change are indicated.

Figure 3

The unusual conformation of ATP seen in GlmU^Mtb[GlcNAc-1-P:ATP]. (a) The regular octahedral hexacoordination goemetry of the three metal ions, Mg_A ²⁺, Mg_B′ ²⁺ and Mg_C ²⁺, is shown. Interactions made by Mg_B′ ²⁺ and Mg_C ²⁺ appear important in positioning Pα away from the nucleophile. (b) Substrates bound at the active site of GlmU^Mtb[GlcNAc-1-P:UTP] (reported in Jagtap et al., 2013 ▶), which is the catalytically active form. UTP and GlcNAc-1-P are shown in sticks with C atoms coloured yellow. An asterisk indicates the attacking nucleophile of GlcNAc-1-P. Magnesium ions bound to both structures are shown as green spheres. Coordination interactions of Mg_B ²⁺ with the triphosphate of UTP are shown. (c) GlmU^Mtb[GlcNAc-1-P:ATP] is in a catalytically inactive state. A superposition with the active GlmU^Mtb[GlcNAc-1-P:UTP] is shown. Nucleotides are marked and shown as sticks with C atoms coloured cyan for ATP and yellow for UTP. In the unusual ATP conformation, Pα (marked Pα-A) is displaced away from the attacking nucleophile of GlcNAc-1-P. The Pα of UTP (marked Pα-U) is in a catalytically competent position optimal for nucleophilic attack. Magnesium ions bound to both structures are shown as spheres; those in dark green correspond to those of GlmU^Mtb[GlcNAc-1-P:ATP] and those in light green correspond to those of GlmU^Mtb[GlcNAc-1-P:UTP]. (d) ATP bound in an atypical comformation at the active site is shown as sticks with C atoms coloured cyan. Residues making interactions with ATP are shown in stick representation; those forming the hydrophobic pocket are shown with C atoms coloured green. Residues Thr18 and Arg19 that interact with the γ-­phosphate are shown in sticks with their C atoms coloured yellow. The hydrogen-bond interaction made by Gln83 with the ribose sugar is shown as a blue broken line. Ala14, Pro16, His58 and Pro86 forming a hydrophobic pocket around the adenine base are shown in sticks with their C atoms coloured green. (e) With the natural substrate, i.e. UTP, Gln83 interacts with the uracil base. UDP-GlcNAc and pyrophosphate at the active site of GlmU^Mtb (PDB entry 4g87) are shown as sticks with their C atoms coloured cyan. Hydrogen-bond interactions of Gln83 with the uracil base are shown as blue broken lines. Residues Thr18 and Arg19 that interact with the γ-­phosphate are shown in sticks with their C atoms coloured yellow. (f) As (c) but in a different orientation. Here, the magnesium ions are labelled. Mg_B′ ²⁺ in GlmU^Mtb[GlcNAc-1-P:ATP] is displaced by 2.1 Å from the corresponding Mg_B ²⁺ in GlmU^Mtb[GlcNAc-1-P:UTP].

Figure 4

A displaced Mg_B ²⁺ is concurrent with the structure of an inactive RNA polymerase. Superposition of the structures of RNA polymerase determined at low (PDB entry 2nvz) and high Mg²⁺ concentrations (PDB entry 2yu9) in complex with UTP (Wang et al., 2006 ▶). C atoms of UTP are coloured purple in the latter and yellow in the former (also indicated as high and low). Similarly, the corresponding Mg²⁺ ions are shown as light (low concentration) and dark (high concentration) green spheres. The two metal ions, Mg_A ²⁺ and Mg_B ²⁺, are indicated and the displacement of Mg_B ²⁺ is shown by a dashed line.