Molecular and Kinetic Characteristics of wild type and mutant Porphobilinogen deaminase

The purpose of this dissertation was to provide an overview of acute intermittent porphyria, focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD), as well as experimentally demonstrating the use of kinetic, structural and thermodynamic approaches to shed light on the enzyme reaction. The key focus was to investigate the effect of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme. Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme

Molecular and Kinetic Characterisation of wild type and mutant Porphobilinogen Deaminase

The purpose of this dissertation was to provide an overview of acute intermittent porphyria, focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD), as well as experimentally demonstrating the use of kinetic, structural and thermodynamic approaches to shed light on the enzyme reaction. The key focus was to investigate the effect of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme. Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme

Heme is a ubiquitous and essential iron containing metallo-organic cofactor required for virtually all aerobic life. Heme synthesis is initiated and completed in mitochondria, followed by certain covalent modifications and/or its delivery to apo-hemoproteins residing throughout the cell. While the biochemical aspects of heme biosynthetic reactions are well understood, the trafficking of newly synthesized heme—a highly reactive and inherently toxic compound—and its subsequent delivery to target proteins remain far from clear. In this review, we summarize current knowledge about heme biosynthesis and trafficking within and outside of the mitochondria

Investigating the structural and functional characteristics of coproporphyrin ferrochelatase

Heme is an essential cofactor in most organisms, this includes bacteria and mammals. The coproporphyrin-dependent heme biosynthesis pathway is specific to Gram positive bacteria and was only discovered in 2015. Before this discovery Gram positive bacteria were assumed to synthesise heme using the same heme synthesis pathway as mammals. As a result, the enzymes within this pathway have not been fully characterised and provide novel targets for antibiotics. Experiments were completed to investigate the protein-protein interactions between HemH and HemQ, two consecutive enzymes in the pathway as they are covalently linked in P. acnes. This used SEC and showed that truncations for the P. acnes HemH-Q protein (HemHL and HemQS) interacted 1:1 whereas other non-covalently linked HemH and HemQ proteins didn’t have observable protein-protein interactions. Kinetic investigation of the wildtype B. subtilis and S. aureus HemH proteins with their endogenous substrate (coproporphyrin) was completed as previous kinetic analysis of B. subtilis HemH used analogues of protoporphyrin IX in the kinetic assays. A combination of spectroscopic techniques were utilised and they show that the two proteins are very active and behave in a broadly similar way to each other. Stopped flow fluorescence spectroscopy proved a useful tool for in depth kinetic investigation into the enzymatic mechanism of coproporphyrin ferrochelatase. Rates constants for enzyme/porphyrin isomerisation, metal chelation and binding constants for substrate binding were estimated using this technique. Finally, functionally important B. subtilis HemH active site mutants (K87A, H88A, E264A/Q) were characterised using similar techniques described for wildtype characterisation. K87A/H88A and E264A/Q are on the non-conserved and conserved active site face, respectively. This shows that whilst K87A, E264A/Q are relatively inactive they are capable of binding coproporphyrin and activity is diminished after binding. H88A has activity comparable to wildtype with weakened coproporphyrin binding and better at iron binding and chelation. This research could provide details for rational drug design of antibiotics that specifically target Gram positive bacteria

Promotion of protoporphyrin IX (PpIX) fluorescence by MEK inhibition in animal models of cancer

Background: Protoporphyrin IX (PpIX) is an endogenous fluorescence that is accumulated in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). The cancer-specific fluorescence of PpIX is used to distinguish tumor from normal tissue, which has proven clinically useful during fluorescence-image guided surgery. This study sought to investigate whether modulation of oncogenic Ras/MEK pathway mediates PpIX accumulation in vitro and in vivo. Methods: For in vitro studies, human breast, lung and prostate cancer cells were treated with or without the MEK inhibitor (U0126) for 20 hours, followed with 5- ALA for 5 hours. The cells were lysed, and the PpIX fluorescence accumulated in the cells was measured with a microplate fluorescence reader (λex 405 nm; λem635 nm). For in vivo studies, the 4T1 mouse breast cancer cells were injected into the right hind flank of 8 week-old Balb/c female mice. U0126 (50 μM/Kg) was injected intratumourally to 4T1 breast tumors for 5/8 h followed by 5- ALA injection. Mice bearing 4T1 breast tumors were also injected intraperitoneally with U0126 for 5 or 8. Furthermore, similar experiments were conducted using a HRAS transgenic mice, which develop spontaneous mammary tumors within 3 months old. Results: In vitro, MEK inhibition increases PpIX fluorescence in human cancer cell lines, but not in normal cell lines. The promotion of PpIX fluorescence by MEK inhibition was most commonly observed among human breast cancer cells. In vivo, PpIX accumulation was observed significantly more in the tumors from mice treated with U0126 and 5-ALA (2-3 fold) than those from mice with vehicle control (DMSO/saline) and 5-ALA. Conclusions: In vitro and in vivo treatment of the MEK inhibitor significantly increases the accumulation of PpIX fluorescence in cancer cells which may contribute to improved efficacy of FGS in clinical settings

Structural and Functional Studies of the Light-Dependent Protochlorophyllide Oxidoreductase Enzyme

The light dependent enzyme protochlorophyllide oxidoreductase (POR) is a key enzyme in the chlorophyll biosynthesis pathway, catalysing the reduction of the C17 C18 bond in protochlorophyllide (Pchlide) to form chlorophyllide (Chlide). This reaction involves the light-induced transfer of a hydride from the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor, followed by proton transfer from a catalytic tyrosine residue. Much work has been done to elucidate the catalytic mechanism of POR, however little is known about the protein structure. POR isoforms in plants are also notable as components of the prolamellar bodies (PLBs), large paracrystalline structures that are precursors to the thylakoid membranes in mature chloroplasts. Bioinformatics studies have identified a number of proteins, related to POR, which contain similar structural features, leading to the production of a structural model for POR. A unique loop region of POR was shown by EPR to be mobile, with point mutations within this region causing a reduction in enzymatic activity. Production of a 2H, 13C, 15N labelled sample of POR for NMR studies has enabled significant advancement in the understanding of the protein structure. This includes the calculation of backbone torsion angles for the majority of the protein, in addition to the identification of multiple dynamic regions of the protein. The protocol for purification of Pchlide, the substrate for POR, has been significantly improved, providing high quality pigment for study of the POR ternary complex. Various biophysical techniques have been used to study the macromolecular structure of these complexes, indicating the formation of large aggregates of the cyanobacterial enzyme induced by substrate binding, similar to PLBs. This has also led to the identification of ring structures, composed of 5 and 6 monomers of POR, which are likely to be the primary components of the cyanobacterial POR structures

XENOBIOTIC REGULATION OF THE ATP BINDING CASSETTE TRANSPORTER ABCB6 AND ITS SIGNIFICANCE TO HEPATIC HEME HOMEOSTASIS

Heme is indispensable for mammalian life. It is an essential component of numerous heme proteins, with functions including oxygen transport and storage, energy metabolism, drug and steroid metabolism and signal transduction. Under normal physiological conditions intracellular free heme levels are extremely low because increased levels of free heme are cytotoxic and accordingly, heme biosynthesis is tightly regulated. Although, 5-aminolevulinic acid synthase (ALAS) mediated regulation of heme synthesis is considered the key step in heme biosynthesis, recent reports have identified a second regulatory step in heme biosynthesis mediated by the mitochondrial ATP binding cassette transporter b6 (Abcb6). Abcb6 expression is directly related to enhanced de novo porphyrin biosynthesis, and Abcb6 overexpression activates the expression of genes important for heme biosynthesis. Thus, Abcb6 represents a previously unrecognized rate-limiting step in heme biosynthesis. The dissertation outlines the progress made since its initiation in understanding the mechanism(s) that regulate Abcb6 expression and the significance of Abcb6 expression to cellular heme homeostasis. Exposure to therapeutic drugs and environmental contaminants leads to an increase in heme demand to compensate for the increased expression of the heme-dependent cytochrome P450s (P450s) detoxifying enzymes. Cells respond to this increasing heme demand by increasing heme synthesis. Thus, exposure to environmental contaminants serves as an optimal in vivo and in vitro model system to study mechanisms that regulate heme synthesis. In this model, Abcb6 expression was induced in response to exposure to xenobiotics [polyaromatic hydrocarbons (PAHs), 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pregnenolone 16alpha -carbonitrile (PCN)] suggesting a co-ordinate induction of Abcb6 to support the increased heme synthesis. Increased Abcb6 expression in response to cellular heme demands was mediated by the xenobiotic sensing nuclear receptors aryl-hydrocarbon receptor (AhR), the constitutive androstane receptor (CAR), and the pregnane-X receptor (PXR). Exposure to environmental contaminants also leads to the generation of oxidative stress, a primary mechanism by which these compounds cause cellular damage. Cells respond to this increased oxidative stress by activating anti-oxidant defense mechanisms, whose principal components include hemo-proteins (such as catalase, superoxide dismutase, etc). Arsenic, an environmental contaminant and a major hazard following occupational exposure exerts its chronic toxicity through the generation of reactive oxygen species. Of importance, exposure to arsenic also activates the antioxidant defense mechanism. Thus, exposure to arsenic serves as a good model system to evaluate in vivo and in vitro oxidative stress response. In this model system, sodium arsenite induced Abcb6 expression in a dose-dependent manner both in mice fed sodium arsenite in drinking water and in cells exposed to sodium arsenite in vitro. Arsenite-induced Abcb6 expression was transcriptionally regulated but was not mediated by the redox sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). The significance of Abcb6 expression to cellular heme homeostasis under conditions of heme demand was evaluated in vitro by both gain of function (cells engineered to overexpress Abcb6) and loss of function (cells where endogenous Abcb6 expression was knocked down using Abcb6 specific ShRNA) analysis. Loss of Abcb6 expression in these in vitro model systems significantly compromises the ability of cells to respond to increased heme demand and the ability to protect against oxidative stress following exposure to environmental contaminants. To understand the significance of Abcb6 function to heme homeostasis in vivo, we generated mice carrying homozygous deletion of the Abcb6 allele (Abcb6 null mice). Abcb6 null animals appear phenotypically normal with a trend towards decreased hepatic heme levels, although, the decreased heme levels did not appear to be statistically significant. Interestingly however, Abcb6 null mice demonstrate genotypic changes that suggest a role for Abcb6 in lipid and cholesterol homeostasis. Abcb6 null mice have increased fasting serum cholesterol and increased accumulation of androstone metabolites. Abcb6 null mice also show decreased expression and activity of a specific set of P450s suggesting a role for Abcb6 in drug metabolism and disposition. Mitochondrial ABC transporters are difficult to study because of the two-membrane architecture of mitochondria, problems associated with analyzing transport process, and the high abundance of other ATPases and carriers/transporters. Thus, the development of an in vitro system with pure and active protein is a prerequisite toward understanding the mechanistic relationships between ATP binding and hydrolysis and coupling of these events to translocation of substrates across the lipid membranes. Towards this end, we developed an in vitro liposomal transport system with pure and active Abcb6 protein. Reconstitution of Abcb6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate stimulated ATPase activity (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III and (iii) transport kinetics of substrates identified using a HTS assay. In summary, this dissertation provides insight into the mechanisms that regulate Abcb6 expression in response to increasing heme demand and the in vitro significance of Abcb6 to cellular heme homeostasis. Development of the Abcb6-null mice suggests that loss of Abcb6 does not severely affect heme-dependent functions in the liver probably because of the activation of compensatory mechanisms that balance the loss of Abcb6. More interestingly, Abcb6-null mice show a phenotype that is characteristic of the deficiency of a protein that is involved in cholesterol and lipid homeostasis. Development of the Abcb6-null mice and the development of an in vitro system with purified Abcb6 should serve as useful tools to understand the transport function of Abcb6 and its role in normal and patho-physiology

The convoluted history of haem biosynthesis

ABSTRACT: The capacity of haem to transfer electrons, bind diatomic gases, and catalyse various biochemical reactions makes it one of the essential biomolecules on Earth and one that was likely used by the earliest forms of cellular life. Since the description of haem biosynthesis, our understanding of this multi‐step pathway has been almost exclusively derived from a handful of model organisms from narrow taxonomic contexts. Recent advances in genome sequencing and functional studies of diverse and previously neglected groups have led to discoveries of alternative routes of haem biosynthesis that deviate from the ‘classical’ pathway. In this review, we take an evolutionarily broad approach to illuminate the remarkable diversity and adaptability of haem synthesis, from prokaryotes to eukaryotes, showing the range of strategies that organisms employ to obtain and utilise haem. In particular, the complex evolutionary histories of eukaryotes that involve multiple endosymbioses and horizontal gene transfers are reflected in the mosaic origin of numerous metabolic pathways with haem biosynthesis being a striking case. We show how different evolutionary trajectories and distinct life strategies resulted in pronounced tensions and differences in the spatial organisation of the haem biosynthesis pathway, in some cases leading to a complete loss of a haem‐synthesis capacity and, rarely, even loss of a requirement for haem altogether