Human 1-D-myo-inositol-3-phosphate synthase is functional in yeast.
We have cloned, sequenced, and expressed a human cDNA encoding 1-d-myo-inositol-3-phosphate (MIP) synthase (hINO1). The encoded 62-kDa human enzyme converted d-glucose 6-phosphate to 1-d-myo-inositol 3-phosphate, the rate-limiting step for de novo inositol biosynthesis. Activity of the recombinant human MIP synthase purified from Escherichia coli was optimal at pH 8.0 at 37 degrees C and exhibited K(m) values of 0.57 mm and 8 microm for glucose 6-phosphate and NAD(+), respectively. NH(4)(+) and K(+) were better activators than other cations tested (Na(+), Li(+), Mg(2+), Mn(2+)), and Zn(2+) strongly inhibited activity. Expression of the protein in the yeast ino1Delta mutant lacking MIP synthase (ino1Delta/hINO1) complemented the inositol auxotrophy of the mutant and led to inositol excretion. MIP synthase activity and intracellular inositol were decreased about 35 and 25%, respectively, when ino1Delta/hINO1 was grown in the presence of a therapeutically relevant concentration of the anti-bipolar drug valproate (0.6 mm). However, in vitro activity of purified MIP synthase was not inhibited by valproate at this concentration, suggesting that inhibition by the drug is indirect. Because inositol metabolism may play a key role in the etiology and treatment of bipolar illness, functional conservation of the key enzyme in inositol biosynthesis underscores the power of the yeast model in studies of this disorder.
Ju
Shulin
S
Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202, USA.
Shaltiel
Galit
G
Shamir
Alon
A
Agam
Galila
G
Greenberg
Miriam L
ML
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002412:Cations; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000737:chemistry; D019753:Intramolecular Lyases / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors; D014635:Valproic Acid / Q000494:pharmacology
D002412:Cations; D018076:DNA, Complementary; D011994:Recombinant Proteins; D007294:Inositol; D014635:Valproic Acid; D019753:Intramolecular Lyases; C105497:D-myo-inositol-3-phosphate synthase