The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid

J Biol Chem. 2017 Nov 10;292(45):18660-18671. doi: 10.1074/jbc.M117.812545. Epub 2017 Sep 14.

Abstract

The periplasmic conversion of ferribactin to pyoverdine is essential for siderophore biogenesis in fluorescent pseudomonads, such as pathogenic Pseudomonas aeruginosa or plant growth-promoting Pseudomonas fluorescens The non-ribosomal peptide ferribactin undergoes cyclizations and oxidations that result in the fluorophore, and a strictly conserved fluorophore-bound glutamic acid residue is converted to a range of variants, including succinamide, succinic acid, and α-ketoglutaric acid residues. We recently discovered that the pyridoxal phosphate-containing enzyme PvdN is responsible for the generation of the succinamide, which can be hydrolyzed to succinic acid. Based on this, a distinct unknown enzyme was postulated to be responsible for the conversion of the glutamic acid to α-ketoglutaric acid. Here we report the identification and characterization of this enzyme in P. fluorescens strain A506. In silico analyses indicated a periplasmic transaminase in fluorescent pseudomonads and other proteobacteria that we termed PtaA for "periplasmic transaminase A" An in-frame-deleted ptaA mutant selectively lacked the α-ketoglutaric acid form of pyoverdine, and recombinant PtaA complemented this phenotype. The ptaA/pvdN double mutant produced exclusively the glutamic acid form of pyoverdine. PtaA is homodimeric and contains a pyridoxal phosphate cofactor. Mutation of the active-site lysine abolished PtaA activity and affected folding as well as Tat-dependent transport of the enzyme. In pseudomonads, the occurrence of ptaA correlates with the occurrence of α-ketoglutaric acid forms of pyoverdines. As this enzyme is not restricted to pyoverdine-producing bacteria, its catalysis of periplasmic transaminations is most likely a general tool for specific biosynthetic pathways.

Keywords: Pseudomonas; enzyme; iron; periplasmic tailoring; pyoverdines; pyridoxal phosphate; siderophore; transaminase.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Coenzymes / metabolism
  • Computational Biology
  • Conserved Sequence
  • Dimerization
  • Expert Systems
  • Gene Deletion
  • Glutamic Acid / metabolism
  • Ketoglutaric Acids / metabolism*
  • Oligopeptides / metabolism*
  • Peptides, Cyclic / metabolism
  • Periplasmic Proteins / chemistry
  • Periplasmic Proteins / genetics
  • Periplasmic Proteins / metabolism*
  • Point Mutation
  • Protein Folding
  • Pseudomonas fluorescens / enzymology*
  • Pseudomonas fluorescens / metabolism
  • Pyridoxal Phosphate / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Transaminases / chemistry
  • Transaminases / genetics
  • Transaminases / metabolism*

Substances

  • Bacterial Proteins
  • Coenzymes
  • Ketoglutaric Acids
  • Oligopeptides
  • Peptides, Cyclic
  • Periplasmic Proteins
  • Recombinant Proteins
  • ferribactin
  • desferriferribactin
  • Glutamic Acid
  • Pyridoxal Phosphate
  • pyoverdin
  • Transaminases

Associated data

  • PDB/3LY1