TY - JOUR
T1 - Human 1-D-myo-inositol-3-phosphate synthase is functional in yeast
AU - Ju, Shulin
AU - Shaltiel, Galit
AU - Shamir, Alon
AU - Agam, Galila
AU - Greenberg, Miriam L.
PY - 2004/5/21
Y1 - 2004/5/21
N2 - We have cloned, sequenced, and expressed a human cDNA encoding 1-D-myo-inositol-3-phosphate (MIP) synthase (hIN01). 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 °C and exhibited Km values of 0.57 mM and 8 μM for glucose 6-phosphate and NAD+, respectively. NH4 + and K+ were better activators than other cations tested (Na+, Li+, Mg2+, Mn2+), and Zn 2+ strongly inhibited activity. Expression of the protein in the yeast ino1Δ mutant lacking MIP synthase (ino1Δ/hIN01) 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 ino1Δ/hIN01 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.
AB - We have cloned, sequenced, and expressed a human cDNA encoding 1-D-myo-inositol-3-phosphate (MIP) synthase (hIN01). 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 °C and exhibited Km values of 0.57 mM and 8 μM for glucose 6-phosphate and NAD+, respectively. NH4 + and K+ were better activators than other cations tested (Na+, Li+, Mg2+, Mn2+), and Zn 2+ strongly inhibited activity. Expression of the protein in the yeast ino1Δ mutant lacking MIP synthase (ino1Δ/hIN01) 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 ino1Δ/hIN01 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.
UR - http://www.scopus.com/inward/record.url?scp=2542422679&partnerID=8YFLogxK
U2 - 10.1074/jbc.M312078200
DO - 10.1074/jbc.M312078200
M3 - Article
C2 - 15024000
AN - SCOPUS:2542422679
SN - 0021-9258
VL - 279
SP - 21759
EP - 21765
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 21
ER -