Cloning and expression of the inositol monophosphatase gene from Methanococcus jannaschii and characterization of the enzyme.
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.
Chen
L
L
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Roberts
M F
MF
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics*; D006358:Hot Temperature; D008094:Lithium / Q000494:pharmacology; D017017:Methanococcus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D019843:Archaeal Proteins; D008094:Lithium; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase