title: Structural and functional analysis of the pyocyanin biosynthetic protein PhzM from Pseudomonas aeruginosa.
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.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA. parsonsj@umbi.umd.edu
authors.forename: James F
authors.initials: JF
authors.lastname: Parsons
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Greenhagen
authors.forename: Katherine
authors.initials: K
authors.lastname: Shi
authors.forename: Kelly
authors.initials: K
authors.lastname: Calabrese
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.forename: Jane E
authors.initials: JE
authors.lastname: Ladner
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D008780:Methyltransferases / Q000737:chemistry* / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000378:metabolism*; D011710:Pyocyanine / Q000096:biosynthesis* / Q000138:chemical synthesis; D012996:Solutions
chemical_list: D001426:Bacterial Proteins; D012996:Solutions; D011710:Pyocyanine; D006899:Mixed Function Oxygenases; C439053:PhzS protein, Pseudomonas aeruginosa; D008780:Methyltransferases; C439052:PhzM protein, Pseudomonas aeruginosa