Publications

• Homologues of neisserial heme oxygenase in Gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa.

  Ratliff M., Zhu W., Deshmukh R., Wilks A., Stojiljkovic I.

  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%) wit ... >> More

  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. << Less

  J. Bacteriol. 183:6394-6403(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Oxidation of heme to beta- and delta-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme.

  Caignan G.A., Deshmukh R., Wilks A., Zeng Y., Huang H.W., Moenne-Loccoz P., Bunce R.A., Eastman M.A., Rivera M.

  The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to delta-biliverdin (70%) and beta-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by (1)H NMR, (13)C NMR, and resonance Raman spectroscopies. Where ... >> More

  The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to delta-biliverdin (70%) and beta-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by (1)H NMR, (13)C NMR, and resonance Raman spectroscopies. Whereas resonance Raman indicates that the heme-iron ligation in pa-HO is homologous to that observed in previously studied alpha-hydroxylating heme oxygenases, the NMR spectroscopic studies suggest that the heme in this enzyme is seated in a manner that is distinct from that observed for all other alpha-hydroxylating heme oxygenase enzymes for which a structure is known. In pa-HO, the heme is rotated in-plane approximately 110 degrees, so the delta-meso-carbon of the major orientational isomer is located within the HO-fold in the place where the alpha-hydroxylating enzymes typically place the alpha-meso-carbon. The unusual heme seating displayed by pa-HO places the heme propionates so that these groups point in the direction of the solvent-exposed heme edge and appears to originate in large part from the absence of stabilizing interactions between the polypeptide and the heme propionates, which are typically found in alpha-hydroxylating heme oxygenase enzymes. These interactions typically involve Lys-16 and Tyr-112, in Neisseriae meningitidis HO, and Lys-16 and Tyr-134, in human and rat HO-1. The corresponding residues in pa-HO are Asn-19 and Phe-117, respectively. In agreement with this hypothesis, we found that the Asn-19 Lys/Phe-117 Tyr double mutant of pa-HO exists as a mixture of molecules exhibiting two distinct heme seatings; one seating is identical to that exhibited by wild-type pa-HO, whereas the alternative seating is very similar to that typical of alpha-hydroxylating heme oxygenase enzymes and is related to the wild-type seating by approximately 110 degrees in-plane rotation of the heme. Furthermore, each of these heme seatings in the pa-HO double mutant gives rise to a subset of two heme isomeric orientations that are related to each other by 180 degrees rotation about the alpha-gamma-meso-axis. The coexistence of these molecules in solution, in the proportions suggested by the corresponding area under the peaks in the (1)H NMR spectrum, explains the unusual regioselectivity of heme oxygenation observed with the double mutant, which we found produces alpha- (55%), delta-(35%), and beta-biliverdin (10%). Alpha-biliverdin is obtained by oxidation of the heme seated similar to that of alpha-hydroxylating enzymes, whereas beta- and delta-biliverdin are formed from the oxidation of heme seated as in wild-type pa-HO. << Less

  J. Am. Chem. Soc. 124:14879-14892(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural basis for novel delta-regioselective heme oxygenation in the opportunistic pathogen Pseudomonas aeruginosa.

  Friedman J., Lad L., Li H., Wilks A., Poulos T.L.

  The Gram-negative bacterium Pseudomonas aeruginosa contains a heme oxygenase (pa-HO) that primarily oxygenates the delta-meso heme carbon [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, ... >> More

  The Gram-negative bacterium Pseudomonas aeruginosa contains a heme oxygenase (pa-HO) that primarily oxygenates the delta-meso heme carbon [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. This differs from other previously characterized heme oxygenases, which display regioselectivity for the alpha-meso heme carbon. Here we report the crystal structure of pa-HO at 1.60 A resolution and compare it to the 1.50 A structure of nm-HO from Neisseria meningitidis [Schuller, D. J., Zhu, W., Stojiljkovic, I., Wilks, A., and Poulos, T. L. (2001) Biochemistry 40, 11552-11558]. The crystal structure of pa-HO maintains the same overall fold as other bacterial and mammalian heme oxygenases, including a conserved network of hydrogen-bonded solvent molecules important for dioxygen activation. The novel delta-regioselectivity of heme oxygenation observed by pa-HO is due to the heme being rotated by approximately 100 degrees, which places the delta-meso heme carbon in the same position as the alpha-meso heme carbon in other heme oxygenases. The main interaction in pa-HO that stabilizes the unique heme orientation is a salt bridge between Lys132 and the heme 7-propionate, as well as hydrophobic contacts involving Leu29, Val33, and Phe189 with the heme methyl and vinyl groups. << Less

  Biochemistry 43:5239-5245(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.