Biochemical and structural characterization of an inositol pyrophosphate kinase from a giant virus.

Kinases that synthesize inositol phosphates (IPs) and pyrophosphates (PP-IPs) control numerous biological processes in eukaryotic cells. Herein, we extend this cellular signaling repertoire to viruses. We have biochemically and structurally characterized a minimalist inositol phosphate kinase (i.e., TvIPK) encoded by Terrestrivirus, a nucleocytoplasmic large ("giant") DNA virus (NCLDV). We show that TvIPK can synthesize inositol pyrophosphates from a range of scyllo- and myo-IPs, both in vitro and when expressed in yeast cells. We present multiple crystal structures of enzyme/substrate/nucleotide complexes with individual resolutions from 1.95 to 2.6 Å. We find a heart-shaped ligand binding pocket comprising an array of positively charged and flexible side chains, underlying the observed substrate diversity. A crucial arginine residue in a conserved "G-loop" orients the γ-phosphate of ATP to allow substrate pyrophosphorylation. We highlight additional conserved catalytic and architectural features in TvIPK, and support their importance through site-directed mutagenesis. We propose that NCLDV inositol phosphate kinases may have assisted evolution of inositol pyrophosphate signaling, and we discuss the potential biogeochemical significance of TvIPK in soil niches.

The inositol pyrophosphate metabolism of Dictyostelium discoideum does not regulate inorganic polyphosphate (polyP) synthesis.

Initial studies on the inositol phosphates metabolism were enabled by the social amoeba Dictyostelium discoideum. The abundant amount of inositol hexakisphosphate (IP6 also known as Phytic acid) present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP7 and IP8, possessing one or two high energy phosphoanhydride bonds, respectively. Considering the contemporary growing interest in inositol pyrophosphates, it is surprising that in recent years D. discoideum, has contributed little to our understanding of their metabolism and function. This work fulfils this lacuna, by analysing the ip6k, ppip5k and ip6k-ppip5K amoeba null strains using PAGE, 13C-NMR and CE-MS analysis. Our study reveals an inositol pyrophosphate metabolism more complex than previously thought. The amoeba Ip6k synthesizes the 4/6-IP7 in contrast to the 5-IP7 isomer synthesized by the mammalian homologue. The amoeba Ppip5k synthesizes the same 1/3-IP7 as the mammalian enzyme. In D. discoideum, the ip6k strain possesses residual amounts of IP7. The residual IP7 is also present in the ip6k-ppip5K strain, while the ppip5k single mutant shows a decrease in both IP7 and IP8 levels. This phenotype is in contrast to the increase in IP7 observable in the yeast vip1Δ strain. The presence of IP8 in ppip5k and the presence of IP7 in ip6k-ppip5K indicate the existence of an additional inositol pyrophosphate synthesizing enzyme. Additionally, we investigated the existence of a metabolic relationship between inositol pyrophosphate synthesis and inorganic polyphosphate (polyP) metabolism as observed in yeast. These studies reveal that contrary to the yeast, Ip6k and Ppip5k do not control polyP cellular level in amoeba.

Inositol phosphate kinases in the eukaryote landscape.

Inositol phosphate encompasses a large multifaceted family of signalling molecules that originate from the combinatorial attachment of phosphate groups to the inositol ring. To date, four distinct inositol kinases have been identified, namely, IPK, ITPK, IPPK (IP5-2K), and PPIP5K. Although, ITPKs have recently been identified in archaea, eukaryotes have taken advantage of these enzymes to create a sophisticated signalling network based on inositol phosphates. However, it remains largely elusive what fundamental biochemical principles control the signalling cascade. Here, we present an evolutionary approach to understand the development of the 'inositol phosphate code' in eukaryotes. Distribution analyses of these four inositol kinase groups throughout the eukaryotic landscape reveal the loss of either ITPK, or of PPIP5K proteins in several species. Surprisingly, the loss of IPPK, an enzyme thought to catalyse the rate limiting step of IP6 (phytic acid) synthesis, was also recorded. Furthermore, this study highlights a noteworthy difference between animal (metazoan) and plant (archaeplastida) lineages. While metazoan appears to have a substantial amplification of IPK enzymes, archaeplastida genomes show a considerable increase in ITPK members. Differential evolution of IPK and ITPK between plant and animal lineage is likely reflective of converging functional adaptation of these two types of inositol kinases. Since, the IPK family comprises three sub-types IPMK, IP6K, and IP3-3K each with dedicated enzymatic specificity in metazoan, we propose that the amplified ITPK group in plant could be classified in sub-types with distinct enzymology.

Microbial inositol polyphosphate metabolic pathway as drug development target.

Inositol polyphosphates are a diverse and multifaceted class of intracellular messengers omnipresent in eukaryotic cells. These water-soluble molecules regulate many aspects of fundamental cell physiology. Removing this metabolic pathway is deleterious: inositol phosphate kinase null mutations can result in lethality or substantial growth phenotypes. Inositol polyphosphate synthesis occurs through the actions of a set of kinases that phosphorylate phospholipase-generated IP3 to higher phosphorylated forms, such as the fully phosphorylated IP6 and the inositol pyrophosphates IP7 and IP8. Unicellular organisms have a reduced array of the kinases for synthesis of higher phosphorylated inositol polyphosphates, while human cells possess two metabolic routes to IP6. The enzymes responsible for inositol polyphosphate synthesis have been identified in all eukaryote genomes, although their amino acid sequence homology is often barely detectable by common search algorithms. Homology between human and microbial inositol phosphate kinases is restricted to a few catalytically important residues. Recent studies of the inositol phosphate metabolic pathways in pathogenic fungi (Cryptococcus neoformans) and protozoa (Trypanosome brucei) have revealed the importance of the highly phosphorylated inositol polyphosphates to the fitness and thus virulence of these pathogens. Given this, identification of inositol kinase inhibitors specifically targeting the kinases of pathogenic microorganisms is desirable and achievable.