Crystal Structure of Archaeoglobus fulgidus CTP:Inositol-1-Phosphate Cytidylyltransferase, a Key Enzyme for Di- myo -Inositol-Phosphate Synthesis in (Hyper)Thermophiles

Abstract

Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3-phosphate-1-phosphate from CDP-inositol and L-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K m values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V max determined at 90°C was 62.9 mol ⅐ min ؊1 ⅐ mg of protein ؊1 . The structure of IPCT is composed of a central seven-stranded mixed ␤-sheet, of which six ␤-strands are parallel, surrounded by six ␣-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X 4 -P-K. CTP, L-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.Enzymes classified in the nucleoside triphosphate-transferase family (PF00483) typically transfer nucleoside monophosphate (NMP) from nucleoside triphosphates (NTP) to an acceptor phosphoryl group belonging to a small molecule such as phosphocholine, hexose-1-phosphate, or ribitol-5-phosphate (2,16,20). This activity leads to release of pyrophosphate and production of a nucleoside diphospho-acceptor that is subsequently utilized by glycosyltransferases in a myriad of reactions of vital importance for the cellular functions.Recently, we described a novel nucleotidyltransferase that uses CTP and L-myo-inositol-1-phosphate (inositol-1P) as substrates to synthesize a newly identified metabolite, CDP-inositol. Additionally, we showed that the inositol group of this metabolite is subsequently transferred to a second molecule of L-myo-inositol-1-phosphate to yield the phosphorylated form of di-myo-inositol phosphate (DIPP), which is then dephosphorylated to give di-myo-inositol phosphate (DIP) (Fig. 1) (31). This phosphodiester of myo-inositol is a hallmark of marine microorganisms that thrive optimally in very hot environments. Indeed, this unusual compatible solute is highly restricted to hyperthermophilic Archaea and Bacteria and has never been found in mesophilic organisms; furthermore, the DIP pool increa...

Structural basis for catalysis at the membrane-water interface

“…These results are in great part shared with those reported concomitantly on di- myo -inositol-1,3′-phosphate-1′-phosphate synthase (DIPPS), a CDP-AP related to Af 2299 and also from Archaeoglobus fulgidus [6]. Interestingly, both Af 2299 and DIPPS carry a soluble N-terminal cytidyltransferase-like domain [8], which provided the essential contacts in the crystal lattice between membrane layers of the lipidic crystallization matrix (lipidic cubic phase; LCP) [6, 7]. …”

X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism

“…1). The structure was solved by molecular replacement using the coordinates of the IPCT domain we previously reported 16 and refined to a resolution of 2.65 Å with R work /R free values of 24.5% and 30%, respectively ( Table 1). The final model comprises 408 amino-acid residues, 1 magnesium ion, 9 water molecules and 4 lipid fragments.…”

Evolution of the biosynthesis of di‐myo‐inositol phosphate, a marker of adaptation to hot marine environments

“…These sequences exhibit around 20% similarity with IPCTs. In this context, it is interesting that the X‐ray structure of the A. fulgidus IPCT domain shows a high structural identity with glucose‐1‐phosphate thymidylyl‐/uridylyl‐transferases (Brito et al ., 2011). In contrast, alcohol nucleotidyltransferases, such as glycerol‐, ribitol‐ or methylerythritol‐cytidylyltransferase, do not share any structural or sequence similarity with IPCTs.…”

“…Recently, the first three‐dimensional structure of an IPCT domain was resolved by X‐ray. Surprisingly, the structure bears homology with glucose‐1‐phosphate thymidylyl/uridylyl‐transferases and is less related to the cytidylyltransferases used for activation of polyol‐phosphates (Brito et al ., 2011). Regrettably, the structure of DIPPS has not been resolved due to difficulties in obtaining good‐quality crystals of this membranar protein.…”