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Title: Structural Analysis and Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa

Authors:
; ; ; ;  [1]
  1. (Queens)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
FOREIGNOTHER
OSTI Identifier:
1129233
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Mol. Biol.; Journal Volume: 426; Journal Issue: (9) ; 05, 2014
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Lee, Michael J.Y., Schep, Daniel, McLaughlin, Brian, Kaufmann, Martin, and Jia, Zongchao. Structural Analysis and Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa. United States: N. p., 2014. Web. doi:10.1016/j.jmb.2014.02.013.
Lee, Michael J.Y., Schep, Daniel, McLaughlin, Brian, Kaufmann, Martin, & Jia, Zongchao. Structural Analysis and Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa. United States. doi:10.1016/j.jmb.2014.02.013.
Lee, Michael J.Y., Schep, Daniel, McLaughlin, Brian, Kaufmann, Martin, and Jia, Zongchao. Mon . "Structural Analysis and Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa". United States. doi:10.1016/j.jmb.2014.02.013.
@article{osti_1129233,
title = {Structural Analysis and Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa},
author = {Lee, Michael J.Y. and Schep, Daniel and McLaughlin, Brian and Kaufmann, Martin and Jia, Zongchao},
abstractNote = {},
doi = {10.1016/j.jmb.2014.02.013},
journal = {J. Mol. Biol.},
number = (9) ; 05, 2014,
volume = 426,
place = {United States},
year = {Mon Apr 21 00:00:00 EDT 2014},
month = {Mon Apr 21 00:00:00 EDT 2014}
}
  • PhzS, an FAD-dependent monooxygenase that catalyzes a reaction involved in the biosynthesis of the virulence factor pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and seleno-l-methionine-labelled crystals is reported. The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystalsmore » belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 Å, α = γ = 90, β = 110.5°. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 Å. Seleno-l-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 Å, α = γ = 90, β = 110.5° and the diffraction limit is 2.7 Å.« less
  • 2,3-Dihydroxybiphenyl dioxygenase, involved in biphenyl and polychlorinated biphenyl degradation, was purified from cell extracts of polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 and Pseudomonas aeruginas PAO1161 carrying the cloned bphC gene (encoding 2,3-dihydroxybiphenyl dioxygenase). The purified enzyme contained ferrous iron as a prosthetic group. The specific activities decreased with the loss of ferrous iron from the enzyme, and the activity was restored by incubation with ferrous iron in the presence of cysteine. Addition of ferric iron caused the complete inactivation of the enzyme. The molecular weight was estimated to be 250,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band with amore » molecular weight of 31,000, indicating that the enzyme consists of eight identical subunits. The enzyme was specific only for 2,3-dihydroxybiphenyl with a K/sub m/ value of 87 ..mu..M. No significant activity was observed for 3,4-dihydroxybiphenyl, catechol, or 3-methyl- and 4-methylcatechol. The molecular weight, subunit structure, ferrous iron requirement, and NH/sub 2/-terminal sequence (starting with serine up to 12 residues) were the same between the two enzymes obtained from KF707 and PAO1161 (bphC).« less
  • 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 acidmore » 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 {angstrom} 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.« less
  • In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD, and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex withmore » the cofactor pyridoxal 5{prime}-phosphate (PLP) and product UDP-GlcNAc(3NH{sub 2})A as the external aldimine at 1.9 {angstrom} resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the {alpha}-carboxylate moiety of {alpha}-ketoglutarate, is also uniquely positioned to bestow specificity for the 6{double_prime}-carboxyl group of GlcNAc(3NH2)A through a salt bridge. This finding is intriguing because while an analogous basic residue is present in WbpE homologues that do not process 6{double_prime}-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C6{double_prime} atom. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2{double_prime}, C3{double_prime}, and C6{double_prime} positions and provides insight into a novel target for treatment of P. aeruginosa infection.« less