Homologues of neisserial heme oxygenase in gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa.
The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-alpha is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.
Ratliff
M
M
Department of Microbiology and Immunology, Emory School of Medicine, Atlanta, Georgia 30322, USA.
Zhu
W
W
Deshmukh
R
R
Wilks
A
A
Stojiljkovic
I
I
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001664:Biliverdine / Q000378:metabolism; D005816:Genetic Complementation Test; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D009345:Neisseria meningitidis / Q000201:enzymology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D016958:Pseudomonas putida / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001426:Bacterial Proteins; D006418:Heme; D006861:Hydrogen Peroxide; C423452:HemO protein, Neisseria meningitidis; D006419:Heme Oxygenase (Decyclizing); D001664:Biliverdine