Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase.
A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.
Chen
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Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Zhou
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Yang
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Roberts
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MF
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000145:classification*; D019843:Archaeal Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D007296:Myo-Inositol-1-Phosphate Synthase / Q000737:chemistry* / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D000446:Aldehyde-Lyases; D007296:Myo-Inositol-1-Phosphate Synthase