Overexpression, purification, and analysis of complementation behavior of E. coli SuhB protein: comparison with bacterial and archaeal inositol monophosphatases

Biochemistry. 2000 Apr 11;39(14):4145-53. doi: 10.1021/bi992424f.


The E. coli suhB gene product, which has been suggested to participate in posttranscriptional control of gene expression, also possesses inositol-1-phosphatase (I-1-Pase) activity. To test if SuhB I-1-Pase activity is sufficient for its function in cells, we have cloned the genes for three other I-1-Pases (from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from the bacterium Thermotoga maritima) into the E. coli expression vector pET23a(+) and examined if these extragenic I-1-Pases could complement the suhB mutation in E. coli strain CG1307 (which also has a mutation in dnaB and a cold-sensitive phenotype). None of these I-1-Pase genes restored growth at 30 degrees C although they generated active I-1-Pase enzymes (as measured by I-1-Pase specific activities of crude protein extracts from the transformed CG1307 cells). In contrast, the pET23a(+) recombinant plasmid with the wild-type E. coli suhB gene complemented the cold sensitivity of the chromosomal mutant suhB and restored the temperature-sensitive growth of the dnaB mutation in the double mutant strain CG1307. Further evidence that this relief of the suppressor behavior of the suhB mutation is not related to the I-1-Pase activity of the SuhB protein was provided by construction of the E. coli SuhB mutant D87N. This mutant protein is inactive as an I-1-Pase but fully functional in changing the temperature sensitivity of the E. coli double mutant strain. Therefore, I-1-P phosphatase activity is neither sufficient nor required for complementation of suhB mutant suppressor effects. The wild-type E. coli SuhB protein was also overexpressed to very high levels and purified to homogeneity in high yield (1 mg/10 mL of culture). The major differences of the E. coli I-1-Pase from all the other characterized I-1-Pases are that it exists as a monomer (rather than a dimer or tetramer) in solution and is more hydrophobic. These physical differences, rather than the I-1-Pase activity, may be involved in the biological role of wild-type SuhB in E. coli.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Molecular Sequence Data
  • Mutation
  • Phosphoric Monoester Hydrolases* / analysis
  • Phosphoric Monoester Hydrolases* / genetics
  • Phosphoric Monoester Hydrolases* / isolation & purification
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Species Specificity


  • Archaeal Proteins
  • Recombinant Proteins
  • Phosphoric Monoester Hydrolases
  • myo-inositol-1 (or 4)-monophosphatase