N-Acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. AGM1 catalyzes the conversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) into N-acetylglucosamine 1-phosphate (GlcNAc-1-P). This enzyme is a member of the ␣-D-phosphohexomutase superfamily, which catalyzes the intramolecular phosphoryl transfer of sugar substrates. Here we report the crystal structures of AGM1 from Candida albicans for the first time, both in the apoform and in the complex forms with the substrate and the product, and discuss its catalytic mechanism. The structure of AGM1 consists of four domains, of which three domains have essentially the same fold. The overall structure is similar to those of phosphohexomutases; however, there are two additional ␤-strands in domain 4, and a circular permutation occurs in domain 1. The catalytic cleft is formed by four loops from each domain. The N-acetyl group of the substrate is recognized by Val-370 and Asn-389 in domain 3, from which the substrate specificity arises. By comparing the substrate and product complexes, it is suggested that the substrate rotates about 180°on the axis linking C-4 and the midpoint of the C-5-O-5 bond in the reaction.

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 ␣-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-GlcNAc 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 ␣-and ␥-phosphate group of UTP.

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-soughtafter inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an alternating access mechanism for the MATE family transporters. multidrug resistance | membrane protein structure | MATE transporter | inward-facing conformation | lipids

Edited by Richard CogdellKeywords: Non-mevalonate pathway E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase Iron-sulfur cluster X-ray structure Drug design a b s t r a c t Isoprenoids are biosynthesized via the mevalonate or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways the latter being used by most pathogenic bacteria, some parasitic protozoa, plant plastids, but not by animals. We determined the X-ray structure of the homodimeric [4Fe-4S] cluster carrying E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) of Thermus thermophilus which catalyzes the penultimate reaction of the MEP pathway and is therefore an attractive target for drug development. The [4Fe-4S] cluster ligated to three cysteines and one glutamate is encapsulated at the intersubunit interface. The substrate binding site lies in front of an (ab) 8 barrel. The great [4Fe-4S] cluster-substrate distance implicates large-scale domain rearrangements during the reaction cycle.Structured summary: gcpE binds to gcpE by x-ray crystallography (View interaction)

The crystal structure of a calcium-free ␣-amylase (AmyK38) from Bacillus sp. strain KSM-K38, which resists chelating reagents and chemical oxidants, has been determined by the molecular replacement method and refined to a crystallographic R-factor of 19.9% (R-free of 23.2%) at 2.13-Å resolution. The main chain folding of AmyK38 is almost homologous to that of Bacillus licheniformis ␣-amylase. However, neither a highly conserved calcium ion, which is located at the interface between domains A and B, nor any other calcium ions appear to exist in the AmyK38 molecule, although three sodium ions were found, one of which is located at the position corresponding to that of a highly conserved calcium ion of other ␣-amylases. The existence of these sodium ions was crystallographically confirmed by the structures of three metal-exchanged and mutated enzymes. This is the first case in which the structure of the calcium-free ␣-amylase has been determined by crystallography, and it was suggested that these sodium ions, instead of calcium ions, are used to retain the structure and function of AmyK38.