Recombinant Production of Biliverdin IXβ and δ Isomers in the T7 Promoter Compatible Escherichia coli Nissle

The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.

Neutron Structure of Phycocyanobilin:oxidoreductase Complexed with Biliverdin

Phytobilins are linear tetrapyrrole compounds used as chromophore for light harvesting and photoreceptor proteins in higher plants, algae, and cyanobacteria. Phytobilins are synthesized from biliverdin IX(alpha) (BV). Phycocianobilin:oxidoreductase (PcyA) is an enzyme to produce phycocyanobilin (PCB) used as chromophore for light harvesting and photoreceptor proteins. PcyA is unique because it catalyzes the reduction of BV by two sequential steps; the first step is the reduction of the vinyl of the BV D-ring to produce 18(1)-18(2)-dihydrobiliverdin (18EtBV), and the second step is the reduction of the A-ring. In these reduction steps, four hydrogen atoms are delivered to BV. The earlier studies showed that the carboxyl group of Asp105 showed dual conformations. This has been attributed to the difference of its protonation states. The catalytically essential His88 was suggested to be protonated (i. e. His88 is a proton donor) to donate the proton to BV. BVH+ (N-protonated) state, in which four pyrrole N atoms of BV were fully protonated, was proposed to be partially formed when BV was bound to PcyA. Further, another tautomeric BVH+ state in which three of four pyrrole N atoms of BV were protonated and the lactam (C=O) group of BV D-ring was protonated as lactim (C-OH; O-protonated) was proposed. Additionally, newly identified water molecule near BV has been suggested to be a proton donor. To elucidate the H atom positions of these molecules, we determined the neutron crystal structure of the PcyA-BV complex at 1.95 Å resolution. Crystal with approximately 2.2 X 1.8 X 0.8 mm3 size, which was soaked into the deuterium-exchanged crystallization solution, was used in the diffraction experiment. The neutron diffraction intensity data was collected using IBARAKI Biological Crystal Diffractometer (iBIX) in J-PARC. In this conference, we report the protonation states of catalytically important residues and BV as well as orientations of water molecules in the PcyA-BV complex.

Therapeutic Potential of Heme Oxygenase-1/Carbon Monoxide in Lung Disease

Heme oxygenase (HO), a catabolic enzyme, provides the rate-limiting step in the oxidative breakdown of heme, to generate carbon monoxide (CO), iron, and biliverdin-IXα. Induction of the inducible form, HO-1, in tissues is generally regarded as a protective mechanism. Over the last decade, considerable progress has been made in defining the therapeutic potential of HO-1 in a number of preclinical models of lung tissue injury and disease. Likewise, tissue-protective effects of CO, when applied at low concentration, have been observed in many of these models. Recent studies have expanded this concept to include chemical CO-releasing molecules (CORMs). Collectively, salutary effects of the HO-1/CO system have been demonstrated in lung inflammation/acute lung injury, lung and vascular transplantation, sepsis, and pulmonary hypertension models. The beneficial effects of HO-1/CO are conveyed in part through the inhibition or modulation of inflammatory, apoptotic, and proliferative processes. Recent advances, however, suggest that the regulation of autophagy and the preservation of mitochondrial homeostasis may serve as additional candidate mechanisms. Further preclinical and clinical trials are needed to ascertain the therapeutic potential of HO-1/CO in human clinical disease.

Linear tetrapyrroles as functional pigments in chemistry and biology

1,19,21,24-tetrahydro-1,19-bilindione is the framework of pigments frequently found in nature, which includes biliverdin IX α, phytochromobilin and phycocyanobilin. 1,19-bilindiones have unique features such as (1) photochemical and thermal cis-trans isomerization, (2) excited energy transfer, (3) chiroptical properties due to the cyclic helical conformation, (4) redox activity, (5) coordination to various metals, and (6) reconstitution to proteins. 1,19-bilindione can adopt a number of conformations since it has exocyclic three double bonds and three single bonds that are rotatable thermally and photochemically. In solution, biliverdin and phycocyanobilin adopt a cyclic helical ZZZ, syn, syn, syn conformation, but other conformations are stabilized depending on the experimental conditions and substituents on the bilin framework. The conformational changes in 1,19-bilindiones are related to the biological functions of a photoreceptor protein, phytochrome. Structural and conformational studies of bilindiones are summarized both in solution and in protein. The conformational changes of bilins can be used for other functions such as a chirality sensor. The bilindiones and the zinc complexes of bilindiones can be employed as a chirality sensor due to the helically chiral structure and the dynamics of racemization of enantiomers. In this paper, we discuss the conformational equilibria and dynamics of bilindiones and its implications in photobiology and materials science.

Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of thepigA Gene of Pseudomonas aeruginosa

ABSTRACT The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. ThepigA 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 ofNeisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced withpigA, 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-β. This differs from the previously characterized heme oxygenases in which biliverdin IX-α is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.