Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii

Biochemistry. 1998 Aug 18;37(33):11629-36. doi: 10.1021/bi980540x.

Abstract

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde-Lyases / chemistry*
  • Aldehyde-Lyases / genetics*
  • Aldehyde-Lyases / metabolism
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics*
  • Aspartic Acid / genetics
  • Chromatography, Gel
  • Cross-Linking Reagents
  • Escherichia coli / genetics
  • Fungal Proteins / biosynthesis
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification
  • Methionine / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Pneumocystis / enzymology*
  • Pneumocystis / genetics
  • Protein Conformation*
  • Recombinant Proteins / biosynthesis
  • Ultracentrifugation

Substances

  • Cross-Linking Reagents
  • FasA protein, Pneumocystis
  • FasB protein, Pneumocystis
  • Fungal Proteins
  • Recombinant Proteins
  • Aspartic Acid
  • Methionine
  • Aldehyde-Lyases
  • dihydroneopterin aldolase