IGPD

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Introduction

Histidine is an essential dietary nutrient for animals, synthesized de novo by plants and microorganisms. Therefore, biosynthetic pathways are potential targets for herbicide development. The synthesis of histidine from the precursor phosphoribosyl pyrophosphate is a complex process catalyzed by nine enzymes. Most of the intermediates in this pathway are unstable, which makes the structural and mechanistic studies of the individual steps difficult. Imidazole glycerol-phosphate dehydratase (IGPD) catalyzes the sixth step of the pathway and is responsible for the dehydration of imidazole glycerol phosphate (IGP) to imidazole acetol phosphate. Several IGPD inhibitors have been identified, including derivatives of triazole propyl phosphonic acid. Reactions catalyzed by IGPD may have an unusual molecular mechanism because the leaving C-2 hydrogen of IGP is not acidic. In contrast, the leaving hydrogen in most enzyme-catalyzed dehydration is relatively acidic due to an adjacent carbonyl or imine group.

IGPDs in fungi, plants, archaea and some eubacteria are monofunctional. Other eubacteria encode bifunctional enzymes in which IGPD is fused to the penultimate enzyme in histidine biosynthesis, histidine-phosphate phosphatase. Metal ions are crucial for IGPD catalysis, but the mechanism remains unclear. In the absence of metals, plant and fungal IGPDs are stable inactive trimers. Mn²⁺ induces aggregation into a catalytically active form that appears to be a 24-mer. Preliminary studies indicate that yeast IGPD crystallizes as a 24-mer with molecular octahedral symmetry, consistent with its aggregated state in solution. But the structure could not be determined because the diffraction of the cubic crystals was too poor.

IGPD Fold: Gene Duplication and Unique Arrangement of a Rare Structural Motif

The IGPD polypeptide forms a single domain consisting of a bundle of four α-helices sandwiched between four-stranded β-sheets (β1-β2-β4-β3 and β5-β6-β8-β7). The fold possesses an internal repeat. The half-domain motif includes a rare left-handed crossover. The sequences of residues 1-93 and 94-202 of F. neoformans IGPD are 19% identical. The most conserved features of internal repeat is the (D/N)XHHXXE motifs in loops β3-α1 (residues 69-75) and β8-α4 (residues 165-171). The duplication of sequence and structural elements suggests the presence of gene duplication in the evolution of IGPD. Gene duplication is a recurring theme in histidine biosynthetic enzymes. The enzymes that catalyze the first two steps of the IGPD dehydration reaction, phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase and imidazole glycerol-phosphate synthase, also have internal repeat properties and are thought to function through similar gene duplication and evolved from fusion events. These enzymes are homologs but not related to IGPD.

Based on a topology search of the Protein Data Bank, the structure database did not find a protein with a fold like IGPD. However, some proteins have subdomains with topologies identical to the IGPD half-domains.  This fold was first identified in some nucleic acid binding proteins. Recently the half-domain was found in several other proteins and is a conserved structural feature of the GHMP kinase superfamily. The N-terminal or C-terminal half-domains of IGPD share 6-12% sequence identity with any of these proteins, suggesting a very ancient relationship between them.

Figure 1. Polypeptide fold of IGPD (Sinha, S.C.; et al. 2004)

Quaternary Structure in the Crystal

IGPD crystallized as a trimer, which is its quaternary structure in metal-free solution. Despite the presence of Mn²⁺ in the crystallization solution, trimeric aggregation is consistent with the lack of bound Mn²⁺ in the crystal structure. Due to the large size and hydrophobic character of the interface, we deduce that the crystalline trimer corresponds to the solution trimer. About 15% of the surface area of each monomer is buried in the subunit interface. The inner C-terminal β-sheet is more ordered than the outer N-terminal β-sheet due to extensive subunit contacts. The hydrophobic side chains (Leu-179, Tyr-98, Tyr-100, Tyr-102, Ala-103, Pro-104, Leu-109, Val-113, Ile-161, Met-183) and the aliphatic moiety of Arg-182, Lys-96 and Arg-111 form a hydrophobic core between the subunits. The bottom surface of the trimer is positively charged, and the intersubunit contacts at this surface include the side chains of Tyr-98 and Lys-175 and sulfate-mediated salt bridges between Arg-97 of one subunit and Lys-175 and His-53 of another. There is a conical polar depression on the top surface of the trimer, which is filled with 18 ordered water molecules.

Figure 2. IGPD quaternary structure (Sinha, S.C.; et al. 2004)