Abstract
Uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc) is a precursor of the bacterial and fungal cell wall. It is also used in a component of N-linked glycosylation and the glycosylphosphoinositol anchor of eukaryotic proteins. It is synthesized from N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and uridine-5'-triphosphate (UTP) by UDP-GlcNAc pyrophosphorylase (UAP). This is an S(N)2 reaction; the non-esterified oxygen atom of the GlcNAc-1-P phosphate group attacks the alpha-phosphate group of UTP. We determined crystal structures of UAP from Candida albicans (CaUAP1) without any ligands and also complexed with its substrate or with its product. The series of structures in different forms shows the induced fit movements of CaUAP1. Three loops approaching the ligand molecule close the active site when ligand is bound. In addition, Lys-421, instead of the metal ion in prokaryotic UAPs, is coordinated by both phosphate groups of UDP-Glc-NAc and acts as a cofactor. However, a magnesium ion enhances the enzymatic activity of CaUAP1, and thus we propose that the magnesium ion increases the affinity between UTP and the enzyme by coordinating to the alpha- and gamma-phosphate group of UTP.
Highlights
Overall Structure of CaUAP1—We have determined CaUAP1 structures in four different forms, which we designated as apo-like (Complex-1), substrate binding (Complex-2), reaction-completed (Complex-3), and product binding (Complex-4) forms (Table 1)
The increase of the MgCl2 concentration enhanced the amount of produced UDP-GlcNAc (Fig. 6A, lanes 2– 8). Other metal ions such as calcium and zinc show similar enhancement (Fig. 6B). These results suggest that the activity of CaUAP1 depends on the concentration of the magnesium ion and that some metal ions including magnesium enhance the enzyme activity
GlmU, and the acetylation domain of GlmU does not exist in CaUAP1
Summary
CaUAP1 crystallized in tion domain are similar in enzymes of prokaryotes and an apo-like form (Complex-1, see Table 1) was purified in a eukaryotes, and they are homologous to that of the manner that differed slightly from our previously described structures of the SpsA GnT I core (SGC) superfamily Intrudes into the active site of another molecule and contacts 10 mg/ml CaUAP1 with its ligands was prepared in 50 mM the ligand inside (17) These contacts may not be observed in Tris-HCl, pH 7.5, and 1 mM DTT and co-crystallized under the solution, because it is known that this enzyme functions as a condition of 100 mM sodium citrate, pH 5.5– 6.0, 20 –30% (w/v) monomer (17). Preparation conditions for co-crystallization and the obtained crystal state of the complexes for UAP and the substrate or product N-acetylglucosmaine-1-phosphate, uridine-5Ј-triphosphate, and uridine-diphospho-N-acetylglucosamine are abbreviated to GlcNAc-1-P, UTP, and UDP-GlcNAc, respectively
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