Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase

Biochemistry. 2000 Oct 10;39(40):12415-23. doi: 10.1021/bi001517q.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aldehyde-Lyases / chemistry*
  • Aldehyde-Lyases / classification*
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / classification
  • Archaeal Proteins / genetics
  • Archaeal Proteins / isolation & purification
  • Archaeoglobus fulgidus / enzymology*
  • Archaeoglobus fulgidus / genetics
  • Catalysis
  • Enzyme Stability / genetics
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Hot Temperature
  • Kinetics
  • Myo-Inositol-1-Phosphate Synthase / chemistry*
  • Myo-Inositol-1-Phosphate Synthase / classification*
  • Myo-Inositol-1-Phosphate Synthase / genetics
  • Myo-Inositol-1-Phosphate Synthase / isolation & purification
  • Nuclear Magnetic Resonance, Biomolecular
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Spectrometry, Fluorescence

Substances

  • Archaeal Proteins
  • Recombinant Proteins
  • Aldehyde-Lyases
  • Myo-Inositol-1-Phosphate Synthase