Biosynthesis of Di-myo-inositol-1,1'-phosphate, a novel osmolyte in hyperthermophilic archaea

Abstract

Biosynthesis of di-myo-inositol-1,1'-phosphate (DIP) is proposed to occur with myo-inositol and myo-inositol 1-phosphate (I-1-P) used as precursors. Activation of the I-1-P with CTP and condensation of the resultant CDP-inositol (CDP-I) with myo-inositol then generates DIP. The sole known biosynthetic pathway of inositol in all organisms is the conversion of D-glucose-6-phosphate to myo-inositol. This conversion requires two key enzymes: L-I-1-P synthase and I-1-P phosphatase. Enzymatic assays using 31P nuclear magnetic resonance spectroscopy as well as a colorimetric assay for inorganic phosphate have confirmed the occurrence of L-I-1-P synthase and a moderately specific I-1-P phosphatase. The enzymatic reaction that couples CDP-I with myo-inositol to generate DIP has also been detected in Methanococcus igneus. 13C labeling studies with [2,3-13C]pyruvate and [3-13C]pyruvate were used to examine this pathway in M. igneus. Label distribution in DIP was consistent with inositol units formed from glucose-6-phosphate, but the label in the glucose moiety was scrambled via transketolase and transaldolase activities of the pentose phosphate pathway.

FIG. 1

Proposed biosynthetic pathway for DIP showing the four key enzymatic activities. Based on similar transformations in other organisms, cofactors are indicated for several of the steps.

FIG. 2

¹H-coupled ³¹P NMR spectrum (202.3 MHz) of an I-1-P synthase assay mixture containing 5 mM G-6-P, 1 mM NAD⁺, 50 mM Tris acetate (pH 8.2), and 100 μl of 60 to 75% ammonium sulfate protein extract that had been incubated at 85°C for 4 h. Note that only I-1-P is a doublet in the coupled spectrum.

FIG. 3

¹H-decoupled ³¹P spectra (202.3 MHz) for analysis of specific I-1-P phosphatase activities from incubations (55°C, 16 h) of mixtures containing 6 mM MgCl₂, 50 mM Tris acetate (pH 8.0), and the following: 2 mM I-1-P and the 0 to 44% fraction (A), 2 mM I-1-P and the 44 to 85% fraction (B), 2 mM G-6-P and the 0 to 44% fraction (C), and 2 mM G-6-P and the 44 to 85% fraction (D).

FIG. 4

³¹P spectrum (121.4 MHz) for analysis of DIP synthase activity. Fifty microliters of CDP-I stock solution (30 mM), 300 μl of dialyzed cell extract, and 5 mM myo-inositol were incubated at 65°C for 2 h. The insets show expansions of the 3- to 5-ppm region, the phosphate monoester region, and the DIP region at ∼−1 ppm. Each phosphorus resonance in the spectrum is labeled.

FIG. 5

¹H-¹H TOCSY NMR spectrum of DIP partially purified from the DIP synthase assay mixture. The vertical dotted line identifies all six DIP inositol protons in the ring spin system.

FIG. 6

Predicted ¹³C labeling for DIP using [2,3-¹³C]pyruvate: ∗, label from C-3 of pyruvate; ▵, label from C-2 of pyruvate.

FIG. 7

¹H-decoupled ¹³C-NMR spectra of DIP obtained from labeling of M. igneus cultures with [2,3-¹³C]pyruvate (A) and [3-¹³C]pyruvate (B). Identities of the different DIP carbons are indicated by numbers above the resonances. Relative enrichment of DIP carbons in panel B is monitored by the integrated intensities.