Cloning and expression of the inositol monophosphatase gene from Methanococcus jannaschii and characterization of the enzyme

Abstract

Inositol monophosphatase (EC 3.1.3.25) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1'-phosphate, an osmolyte found in hyperthermophilic archaeal. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (K(m) = 0.091 +/- 0.016 mM; Vmax = 9.3 +/- 0.45 mumol of Pi min-1 mg of protein-1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+ inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.

FIG. 1

Proposed DIP biosynthetic pathway. Glucose-6-phosphate is converted to I-1-P (step 1), some of which is hydrolyzed to myo-inositol (step 2), and I-1-P is activated to CDP-I (step 3) for a final reaction in which CDP-I is coupled to myo-inositol (step 4), generating DIP and CMP.

FIG. 2

I-1-Pase expression and purification analyzed by SDS–12% PAGE stained with Coomassie brilliant blue. Lane a, crude extract; lane b, heat-treated crude extract; lane c, purified M. jannaschii I-1-Pase; lanes d and e, molecular mass standards (from the top, 66, 45, 36, 29, 24, 21.4, and 14 kDa).

FIG. 3

Dependence of M. jannaschii I-1-Pase activity (toward 2.5 mM I-1-P in 50 mM Tris HCl [pH 8.0] at 85°C) on Mg²⁺ concentration. The activities are normalized to the value obtained with 10 mM MgCl₂. The error bars indicate standard deviations.

FIG. 4

Effect of Li⁺, Na⁺, and K⁺ on the activity of M. jannaschii I-1-Pase toward 2.5 mM I-1-P in 50 mM Tris HCl, pH 8.0, with 10 mM MgCl₂ at 85°C for 1 min. The activities are normalized to the value for the assay without the monovalent cation salt added. The error bars indicate standard deviations.

FIG. 5

(A) Thermal stability of M. jannaschii I-1-Pase after preincubation at 100°C for various times. The enzyme activity was measured after the preincubation time by adding 0.4 μg of protein to the standard assay mixture and incubating the mixture at 100°C for 1 min. (B) Temperature (T) dependence of M. jannaschii I-1-Pase V_max. The error bars indicate standard deviations.

FIG. 6

Sequence alignment between human and M. jannaschii I-1-Pase (MJ109). The residues discussed in the text are in boldface; dashes represent gaps. A single dot indicates similar residues (polar, nonpolar, etc.), whereas two dots indicate conserved residues.