Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight

J Biol Chem. 2017 Sep 29;292(39):16360-16367. doi: 10.1074/jbc.M117.805028. Epub 2017 Aug 22.

Abstract

5-Oxoproline (OP) is well-known as an enzymatic intermediate in the eukaryotic γ-glutamyl cycle, but it is also an unavoidable damage product formed spontaneously from glutamine and other sources. Eukaryotes metabolize OP via an ATP-dependent 5-oxoprolinase; most prokaryotes lack homologs of this enzyme (and the γ-glutamyl cycle) but are predicted to have some way to dispose of OP if its spontaneous formation in vivo is significant. Comparative analysis of prokaryotic genomes showed that the gene encoding pyroglutamyl peptidase, which removes N-terminal OP residues, clusters in diverse genomes with genes specifying homologs of a fungal lactamase (renamed prokaryotic 5-oxoprolinase A, pxpA) and homologs of allophanate hydrolase subunits (renamed pxpB and pxpC). Inactivation of Bacillus subtilis pxpA, pxpB, or pxpC genes slowed growth, caused OP accumulation in cells and medium, and prevented use of OP as a nitrogen source. Assays of cell lysates showed that ATP-dependent 5-oxoprolinase activity disappeared when pxpA, pxpB, or pxpC was inactivated. 5-Oxoprolinase activity could be reconstituted in vitro by mixing recombinant B. subtilis PxpA, PxpB, and PxpC proteins. In addition, overexpressing Escherichia coli pxpABC genes in E. coli increased 5-oxoprolinase activity in lysates ≥1700-fold. This work shows that OP is a major universal metabolite damage product and that OP disposal systems are common in all domains of life. Furthermore, it illustrates how easily metabolite damage and damage-control systems can be overlooked, even for central metabolites in model organisms.

Keywords: 5-oxoproline; bacterial metabolism; glutamate; glutamine; metabolism; metabolite damage; metabolite repair; microbiology; pyroglutamate.

MeSH terms

  • Allophanate Hydrolase / genetics
  • Allophanate Hydrolase / metabolism*
  • Amidohydrolases / genetics
  • Amidohydrolases / isolation & purification*
  • Amidohydrolases / metabolism
  • Bacillus subtilis / enzymology*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Gene Deletion
  • Gene Knockout Techniques
  • Genomics / methods
  • Multigene Family
  • Mutation
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Pyrrolidonecarboxylic Acid / metabolism
  • Recombinant Proteins / metabolism

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Protein Subunits
  • Recombinant Proteins
  • Amidohydrolases
  • Allophanate Hydrolase
  • 5-oxoprolinase (ATP-hydrolyzing)
  • Pyrrolidonecarboxylic Acid

Associated data

  • PDB/3ORE