Structural and functional analysis of the pyocyanin biosynthetic protein PhzM from Pseudomonas aeruginosa

Biochemistry. 2007 Feb 20;46(7):1821-8. doi: 10.1021/bi6024403. Epub 2007 Jan 25.

Abstract

Pyocyanin is a biologically active phenazine produced by the human pathogen Pseudomonas aeruginosa. It is thought to endow P. aeruginosa with a competitive growth advantage in colonized tissue and is also thought to be a virulence factor in diseases such as cystic fibrosis and AIDS where patients are commonly infected by pathogenic Pseudomonads due to their immunocompromised state. Pyocyanin is also a chemically interesting compound due to its unusual oxidation-reduction activity. Phenazine-1-carboxylic acid, the precursor to the bioactive phenazines, is synthesized from chorismic acid by enzymes encoded in a seven-gene cistron in P. aeruginosa and in other Pseudomonads. Phenzine-1-carboxylic acid is believed to be converted to pyocyanin by the sequential actions of the putative S-adenosylmethionine-dependent N-methyltransferase PhzM and the putative flavin-dependent hydroxylase PhzS. Here we report the 1.8 A crystal structure of PhzM determined by single anomalous dispersion. Unlike many methyltransferases, PhzM is a dimer in solution. The 36 kDa PhzM polypeptide folds into three domains. The C-terminal domain exhibits the alpha/beta-hydrolase fold typical of small molecule methyltransferases. Two smaller N-terminal domains form much of the dimer interface. Structural alignments with known methyltransferases show that PhzM is most similar to the plant O-methyltransferases that are characterized by an unusual intertwined dimer interface. The structure of PhzM contains no ligands, and the active site is open and solvent-exposed when compared to structures of similar enzymes. In vitro experiments using purified PhzM alone demonstrate that it has little or no ability to methylate phenzine-1-carboxylic acid. However, when the putative hydroxylase PhzS is included, pyocyanin is readily produced. This observation suggests that a mechanism has evolved in P. aeruginosa that ensures efficient production of pyocyanin via the prevention of the formation and release of an unstable and potentially deleterious intermediate.

Publication types

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

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / physiology
  • Binding Sites
  • Crystallography, X-Ray
  • Dimerization
  • Methyltransferases / chemistry*
  • Methyltransferases / physiology
  • Mixed Function Oxygenases / chemistry
  • Models, Molecular
  • Protein Conformation
  • Protein Structure, Tertiary
  • Pseudomonas aeruginosa / enzymology
  • Pseudomonas aeruginosa / metabolism*
  • Pyocyanine / biosynthesis*
  • Pyocyanine / chemical synthesis
  • Solutions

Substances

  • Bacterial Proteins
  • Solutions
  • Pyocyanine
  • Mixed Function Oxygenases
  • PhzS protein, Pseudomonas aeruginosa
  • Methyltransferases
  • PhzM protein, Pseudomonas aeruginosa

Associated data

  • PDB/2IP2