Michael Murphy

Professor

Research Classification

Research Interests

Alternatives to antibiotics
Bacterial pathogenesis
Biological and Biochemical Mechanisms
Functional and Structural Proteomics
Microbial metal metabolism

Relevant Degree Programs

Affiliations to Research Centres, Institutes & Clusters

 
 

Research Methodology

Structural biology (crystallography, NMR)
Enzymology
Binding and ligand transfer

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Master's students
Doctoral students
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Any time / year round
I support experiential learning experiences, such as internships and work placements, for my graduate students and Postdocs.
I am open to hosting Visiting International Research Students (non-degree, up to 12 months).
I am interested in hiring Co-op students for research placements.

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2021)
Antimicrobial properties of Kisameet clay, a natural clay mineral from British Columbia, Canada (2019)

The dearth of new antibiotics and the widespread emergence of multi-drug resistant (MDR) bacteria have created a global crisis in medicine and highlighted the drastic need for novel antimicrobial agents. Natural clay minerals with a long history of therapeutic and biomedical applications have recently received increasing attention due to recent studies on their potent antibacterial activity. Kisameet clay (KC), a natural clay deposit found in British Columbia, Canada, has been used by local First Nation people for medicinal purposes for generations. This research investigates the antimicrobial properties of KC, the spectrum of activity, and the mechanism(s) underlying its antibacterial action. In order to characterize KC antimicrobial activity and define its active components, a series of integrated microbiological, chemical, and mineralogical studies have been performed. This study revealed that aqueous suspensions of KC exhibit in vitro broad-spectrum antimicrobial activity against a variety of MDR bacterial pathogens, including the ESKAPE pathogens and Cystic fibrosis clinical isolates. Moreover, two major fungal pathogens, Candida albicans and Cryptococcus neoformans were also susceptible to KC. In addition, KC aqueous leachates (KC-L) show potent bactericidal activity in which low-pH plays a key role. Treatment of KC minerals and KC-L with cation-chelating agents indicates roles for divalent and trivalent cations, more specifically iron and aluminum. Further studies suggest that the bactericidal activity of KC-L is due to multiple modes of action. The low-pH buffered environment, rich in a combination of released metal ions, can synergistically challenge treated bacteria to maintain their metal homeostasis, while aluminum-related impairment of outer membrane (OM) permeability may exacerbate this situation. KC-L can, concurrently, stress multiple bacterial components, cause metal intoxication and consequential cell damage, impair OM and destabilize the cell membrane structure. Furthermore, it induces oxidative stress, generates hydrogen peroxide, and damages DNA, which collectively leads to lethal pleiotropic effects in treated bacteria. Further studies detected KC-L-related transcriptional modulation in oxidative- and envelope- stress responses, DNA damage, metal detoxification pathways, or efflux pump function. Better understanding of the principal components of KC antibacterial activity may permit the formulation of defined, active preparations of this natural clay mineral for therapeutic applications.

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SbnI is a free serine kinase and heme-sensing regulator required for staphyloferrin B biosynthesis in Staphylococcus aureus (2018)

Staphylococcus aureus is a common member of the human microbiome, but is an opportunistic pathogen that can cause a variety of infections. Critical to the growth and survival of S. aureus during infection is acquisition of iron from the host. However, host iron availability is restricted to effectively suppress microbial growth as a type of innate, nutritional immunity. Mechanisms used by S. aureus to access the host iron pool include lysing erythrocytes to liberate hemoglobin for heme uptake and through the secretion of staphyloferrins, which are iron-chelating siderophores to scavenge iron from the host. The multiplicity of iron uptake systems S. aureus possesses is likely a reflection of the varied host environments that S. aureus can colonize or infect. However, the spatiotemporal regulatory mechanisms by which S. aureus adapts to changing iron availability over the course of infection are ill-defined. SbnI is a heme-dependent regulator of staphyloferrin B (SB) biosynthesis suggested to mediate between iron-uptake modes.In this thesis, study of the structure SbnI revealed homology to a free L-serine kinase, SerK, from Thermococcus kodakarensis. Biochemical assays and characterization of a serC mutant of S. aureus showed that SbnI is an ATP dependent L-serine kinase required for production of the SB precursor O-phospho-L-serine. SbnI kinase activity enables SB biosynthesis in environments where S. aureus catabolism is primarily reliant on amino acids, as in abscesses. Characterization of heme binding by SbnI and heme transfer reactions with IsdI and IsdG, two heme degrading enzymes, were used to construct a model of heme-binding by SbnI for regulating heme-SB uptake. This model is consistent with a modest effect of heme-binding on SbnI kinase activity. Heme transfer rates were measured from ChdC, the terminal enzyme in heme biosynthesis, to SbnI, IsdG, and IsdI to delineate an intracellular network of heme sensing and trafficking proteins that are likely required for regulation and adaptation to the dynamic host environment.

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Siderophore-mediated iron metabolism in Staphylococcus aureus (2016)

Staphylococcus aureus requires iron as a nutrient and uses uptake systems to extract iron from the human host. S. aureus produces the iron-chelating siderophore staphyloferrin B (SB) to scavenge for available iron under conditions of low iron stress. Upon iron-siderophore re-entry into the cell, iron is separated from the siderophore complex to initiate assimilation into metabolism. To gain insight into how SB biosynthesis is integrated into S. aureus central metabolism, the three SB precursor biosynthetic proteins, SbnA, SbnB, and SbnG, were biochemically characterized. SbnG is a citrate synthase analogous to the citrate synthase enzyme present in the TCA cycle. The crystal structure of SbnG was solved and superpositions with TCA cycle citrate synthases support a model for convergent evolution in the active site architecture and a conserved catalytic mechanism. Since L-Dap is an essential precursor for SB, the biosynthetic pathway for L-Dap was elucidated. A combination of X-ray crystallography, biochemical assays and biophysical techniques were used to delineate the reaction mechanisms for SbnA and SbnB, demonstrating that SbnA performs a β-replacement reaction using O-phospho-L-serine (OPS) and L-glutamate to produce N-(1-amino-1-carboxy-2-ethyl)-glutamic acid (ACEGA). Oxidative hydrolysis of ACEGA catalyzed by SbnB produces α-ketoglutarate and L-Dap. Detailed analysis of the substrate specificity of SbnA revealed that OPS binding and conversion to the PLP-α-aminoacrylate intermediate in SbnA induced a conformational change and formation of a second substrate binding pocket for L-glutamate. Furthermore, L-cysteine was identified as a competitive inhibitor of SbnA activity, revealing a link between iron uptake and the oxidative stress response in S. aureus. IruO was examined for its role in Fe(III)-siderophore reduction. Utilizing a combination of visible spectroscopy and enzyme kinetics, a mechanism for electron transfer was proposed. IruO was demonstrated to reduce iron bound hydroxamate-type siderophores to release Fe(II) using NADPH as the electron donor. Under anaerobic conditions, IruO formed a stable FAD semiquinone intermediate that mediates a single electron transfer from the FAD to the Fe(III)-siderophore complex. These studies have shown how SB precursors are synthesized and led to the development of models for SB biosynthesis integration into central metabolism under conditions of low iron stress.

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Hemoglobin Binding, Heme Extraction and Heme Transfer by the Staphylococcus aureus Surface Protein IsdB (2014)

Gram-positive Staphylococcus aureus is a common member of the normal human flora, but can also cause serious infections. Survival and growth of S. aureus is dependent on the acquisition of iron from the host, wherein the majority of iron occurs as part of the heme molecule in the oxygen-carrier protein hemoglobin (Hb). S. aureus possesses a system of proteins designed to use heme and hemoglobin as an iron source: the iron-regulated surface determinant (Isd) system. IsdB is the primary Hb receptor and extracts heme from Hb at the cell surface for transfer to IsdA or IsdC, which then transfer it to the membrane transporter for internalization. IsdB contains two NEAT domains (IsdB-N1 and IsdB-N2) which were hypothesized to carry out the Hb-binding, heme binding and heme transfer functions of the protein.Heme binding by IsdB-N2 was characterized biochemically and the crystal structure of heme-reconstituted IsdB-N2 was solved. IsdB-N2 bore the canonical eight-stranded β-sandwich NEAT domain fold and used a conserved Tyr residue to coordinate heme-iron, as well as a non-conserved Met residue, resulting in a novel Tyr-Met hexacoordinate heme-iron. Biochemical differences between equivalent mutations produced in IsdBN² and IsdBN¹N² introduced the possibility of intraprotein domain interactions.The molecular mechanism for heme transfer from IsdB-N2 to IsdA-N1 was investigated using stopped-flow spectroscopy and the kinetics of heme transfer from IsdB-N2 to IsdA-N1 were modeled. The rate of heme transfer between the isolated NEAT domains was similar to that measured for the full-length proteins.Only a recombinant construct with both domains in a contiguous unit (IsdBN¹N²) could bind Hb with high affinity. Spectroscopic analysis demonstrated that both domains were also required to extract heme from Hb. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed heme transfer from Hb to IsdA at a rate 370-fold slower than heme transfer from IsdBN² to IsdAN¹, revealing that heme transfer from Hb to IsdB is the rate-limiting step in this pathway. Finally, the serum Hb-binding protein haptoglobin blocked heme uptake from Hb by IsdB, revealing new areas for exploration of function. These studies provide insight into mechanisms of host-pathogen interactions during infection.

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Iron Binding and Oxidation by Pseudo-nitzschia multiseries ferritin (2014)

A novel ferritin was identified in marine pennate diatoms, unicellular photosynthetic organisms that play a major role in global primary production and carbon sequestration in the deep ocean. The expression of the iron storage and detoxifying protein ferritin is thought to facilitate the blooming of pennate diatoms after iron fertilization in the open ocean.X-ray structures of Pseudo-nitzschia multiseries ferritin (PmFTN) from crystals soaked for various durations in ferrous iron and zinc sulfate revealed three distinct metal binding sites; sites A and B comprise the catalytic ferroxidase centre, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous iron soaked crystals revealed only one ferrous ion occupying site A. Together with kinetic analysis, these studies suggest a model of stepwise iron binding to the ferroxidase centre of PmFTN followed by a very fast iron oxidation phase and partial mobilization of iron from the ferroxidase centre. Using a combination of rapid reaction kinetics and high resolution crystallography, the function of site C was investigated with site C and site B/C variants. Glu130, a site B/C ligand, functions in stabilizing Fe(III) bound at the ferroxidase centre and as a consequence reducing the rate of mineralization. Furthermore, Glu44, a site C ligand, is shown to be important for limiting the rate of post-oxidation reorganisation of iron coordination. Iron was observed within the B-channels, first identified in prokaryotic ferritins and BFRs, of the E44Q variant of PmFTN and provides the first evidence that these channels are possible routes for Fe(II) entry into the cavity. The anaerobic crystal structure of the bacterioferritin from E. coli (EcBFR) revealed two Fe(II) at the ferroxidase centre sites A and B. In comparison with PmFTN, differences in ferrous iron binding and reaction rates are further evidence that in EcBFR a distinct mechanism is in operation.Clearly, PmFTN shows some characteristics of bacterial ferritins. Moreover, retention of iron at the ferroxidase centre at the expense of mineralization points to a role for this diatom ferritin in facilitating short term rather than long term iron storage.

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Iron transport in two pathogenic Gram-negative bacteria (2011)

Campylobacter jejuni and Escherichia coli strain F11 are two Gram-negative pathogens with a versatile armament of iron uptake systems to cope with the fluctuating host nutrient environment. Our current understanding of Gram-negative iron uptake systems focuses heavily on a prototypical scheme involving a TonB-dependent outer membrane receptor and an ABC transporter, with little knowledge on systems that do not fall neatly into this paradigm. The primary focus of this thesis is the characterization of three such atypical iron uptake proteins from C. jejuni (ChaN and P19) and pathogenic E. coli (FetP).C. jejuni ChaN is a 30 kDa, iron-regulated lipoprotein hypothesized to be involved in iron uptake. The crystal structure of ChaN reveals that it can bind two cofacial heme groups in a pocket formed by a ChaN dimer. Each heme iron is coordinated by a single tyrosine from one monomer and the propionate groups are hydrogen bonded by a histidine and a lysine from the other monomer. Analytical ultracentrifugation studies demonstrate heme-dependent dimerization in solution. Cell fractionation of C. jejuni shows that ChaN is localized to the outer membrane. Based on these findings, the predicted in vivo role of ChaN in iron uptake is discussed.C. jejuni cFtr1-P19 and E. coli FetMP are homologous iron-regulated systems also proposed to be iron transporters. Through growth studies in both organisms, we show that P19 and FetMP are required for optimal growth under iron-limited conditions. Furthermore, metal binding analysis demonstrates that recombinant P19 and FetP bind both copper and iron. Dimerization of P19 is shown to be metal dependent in vitro and is detected in vivo by cross-linking. Through x-ray crystallography, we have determined the structures of P19 and FetP with various metals bound, thus revealing the locations of the highly conserved copper and iron binding sites. Additionally, the crystal structure of FetP reveals two copper positions in each binding site that is likely functionally important. Through mutagenesis, residues contributing to the alternative copper positions were identified. Together, these studies provide insight into the mechanism of iron transport by the two systems and allow for the development of functional models.

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Cell surface iron-complex binding and transport by Staphylococcus aureus (2010)

Iron uptake systems are paramount to the survival of many organisms. Pathogenic bacteria are faced with the especially daunting task of acquiring essential iron within their host environment. Staphylococcus aureus is a Gram-positive bacterial pathogen and one of the most common causes of bacterial infections in hospitals. In addition, multi-drug resistant S. aureus isolates are emerging and now constitute the majority of isolated strains from clinical settings. The prevalence of S. aureus is attributed, in part, to its ability to specifically use most host iron sources for growth. S. aureus uses high affinity uptake systems for many different forms of iron in the human body with the source preference varying through the time course of infection and the tissues infected. To gain insight into iron binding and import by S. aureus, surface receptors from the iron surface determinant (Isd) heme uptake system and the staphyloferrin A siderophore uptake systems (unfortunately named heme transfer system (Hts)) were studied. The systems use distinct methods for ligand import. In the Isd system, heme is received and relayed through cell wall anchored proteins (including IsdA) to the substrate binding protein (IsdE) for import through the permease. Crystal structures of IsdA and IsdE in complex with heme, in concert with in vitro heme transfer kinetics contributed to the development of a heme transfer model for NEAT domains. In contrast to heme uptake, staphyloferrin A is bound directly at the substrate binding protein (HtsA). HtsA and IsdE are homologous membrane anchored binding proteins and both receive and deliver the iron-complex to the permease. Crystal structures and ligand affinity measurements of IsdE and HtsA reveal distinct mechanisms for ligand reception and specificity. Furthermore, crystal structures of open and closed conformations of HtsA highlight unique structural changes proposed to enable discrimination by the permease of ligand-bound and -free receptor. These studies provide insight into iron import in S. aureus, which have contributed to the development of models for heme and siderophore transport from the cell surface to the permease.

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Characterization of two new classes of periplasmic ferric binding proteins (2009)

Pathogenic bacteria acquire essential iron using specialized iron acquisitionsystems, such as the FbpABC transport system. The periplasmic FbpA protein deliversiron to the ABC transporter. FbpA proteins have two domains with the iron binding sitelocated at the domain interface. A flexible inter-domain hinge region facilitates substrate dependent conformations. In general, the closed conformations are observed for holo FbpA proteins whereas the apo proteins exhibit increased hinge motion relative to the closed conformation. Closed conformations are likely important for initiating iron translocation across the inner membrane permease. Important bacterial pathogens such as Campylobacter jejuni and Bordetella pertussis contain previously uncharacterized FbpA proteins. Using phylogenetic analyses, six FbpA classes were defined which vary in conservation of iron site ligands and utilization of a synergistic anion. Class I includes theanion-dependent neisserial FbpA (nFbpA). This thesis characterizes the Class III FbpAfrom Campylobacterjejuni (cFbpA) and the Class II FbpA from Bordetella pertussis(bFbpA). Visible spectroscopy showed high affinity iron binding of cFbpA. X-raycrystallography showed anion-independent iron coordination by cFbpA using a histidineand four tyrosine residues. Confonnational analyses in solution by small angle x-rayscattering (SAXS) showed that cFbpA undergoes limited hinge motion in solution upon substrate binding. Furthermore, an iron uptake role is supported as a cJbpA deletionstrain, constructed from C. jejuni 81-176, exhibited impaired growth under iron-limitedconditions. Characterization of bFbpA by visible spectroscopy showed high affinity iron binding with carbonate, citrate and oxalate. Distinct holo conformations compared with the apo conformation were observed for bFbpA depending on the synergistic anion. The closed conformation holo bFbpA crystal structure shows iron coordination by carbonate and three tyrosine residues. SAXS analyses also showed that oxalate and citrate treated holo bFbpA exhibit distinct conformations from apo bFbpA in solution. Furthermore,bFbpA undergoes large hinge motion in solution similar to nFbpA. Models for irontransport are proposed in which these bob complexes of bFbpA and cFbpA arecandidates for initiating productive interactions with the permease.

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Master's Student Supervision (2010 - 2020)
Pseudo-nitzchia multiseries ferritin iron oxidation and oxidative stress protection in the presence of glutathione (2020)

Iron is an essential nutrient for most organisms, including diatoms. A portion of intracellular iron is attributed to the labile iron pool (LIP), which is readily exchangeable iron available to bind to enzymes to participate in biological reactions. Iron belonging to the LIP is transiently bound to cellular ligands, such as glutathione (GSH). However, iron present in the cell in excess can be toxic to the cell, as ferrous iron can react with H₂O₂ in the Fenton reaction to produce highly detrimental reactive oxygen species (ROS). Pseudo-nitzchia multiseries is a marine planktonic diatom that plays an important role in primary production and carbon sequestration in the ocean. P. multiseries expresses an iron storage protein, ferritin (PmFtn), which protects the cell from oxidative damage by oxidizing iron at ferroxidase centres and storing iron in a nano-cage formed from 24 monomers. Ferritin ferroxidase activity is poorly characterized in the presence of biologically-relevant iron chelators of the LIP. In this study, PmFtn ferroxidase activity was found to proceed at a slower rate in the presence of GSH. In a PmFtn structure obtained from a crystal soaked in the presence of iron and GSH for 30 minutes, iron was found bound to the ferroxidase centre at sites A and B, consistent with spectroscopic data showing rapid binding of iron but slow mineralization in the presence of GSH. PmFtn and GSH also protected DNA from H₂O₂ mediated oxidative stress in the presence of iron.

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Investigating the physiological function of a Staphylococcus aureus Ntn-hydrolase (2019)

The N-terminal nucleophile (Ntn)-hydrolase structural superfamily consists of several classes of enzymes, including the penicillin V acylases (PVAs), bile salt hydrolases, and N-acylhomoserine lactone (AHL) acylases. The PVAs use an N-terminal cysteine residue to hydrolyze the amide bond in penicillin V and are most closely related to the bile salt hydrolases. PVAs are encoded by many environmental and pathogenic bacteria and are of great importance in the pharmaceutical industry because the product of penicillin V hydrolysis is used in the production of semi-synthetic antibiotics. Despite their industrial value, the physiological function of the PVAs remains unknown. The opportunistic human pathogen Staphylococcus aureus encodes an Ntn-hydrolase (gene locus SAUSA300_0269). Several potential substrates of SAUSA300_0269 were tested and it was demonstrated to hydrolyze penicillin V but show no activity towards the bile salt glycocholic acid. Based on the observed activity, SAUSA300_0269 was renamed to SaPVA. This enzyme also hydrolyzed several AHLs, which are quorum sensing molecules used by Gram-negative bacteria. SaPVA is the first example of a PVA from a Gram-positive bacterium that cross-reacts with AHLs. The enzyme displayed a preference for unsubstituted AHLs with an acyl chain of six or more carbons. Growth experiments did not support a role for SaPVA in protection of S. aureus against the toxicity of 3-oxo-C12-HSL, an AHL produced by Pseudomonas aeruginosa. Two siderophores used by S. aureus, enterobactin and staphyloferrin A, were also tested as substrates, but SaPVA did not show activity towards either molecule. To obtain further insight into the substrates of SaPVA, the crystal structure was solved to 1.9 Å resolution and compared with those of other characterized PVAs from Gram-positive and Gram-negative bacteria. Similarity in the overall structure and substrate-binding loops of PVAs from Gram-positive bacteria suggest that these enzymes act on similar substrates. Molecular docking was used to predict the binding modes of penicillin V and various AHLs to SaPVA. Docking results provided some clues about the structural features that may be present in physiological substrates of SaPVA, including one or more rings, as well as an aryl group or hydrophobic acyl chain.

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Characterisation of substrate preference in staphylococcus aureus siderophore biosynthesis (2018)

Staphylococcus aureus is a common opportunistic pathogen and commensal resident of a majority proportion of the adult population. Emerging drug-resistant and hypervirulent strains such as methicillin-resistant S. aureus (MRSA) have reduced the efficacy of existing treatment options. Iron acquisition from the host is required for the establishment of infection. S. aureus possesses several mechanisms for iron acquisition, including the ferric-iron binding siderophores staphyloferrin A (SA) and staphyloferrin B (SB). To explore the substrate preference of the SA and SB biosynthetic enzymes, crystal structures of biosynthetic enzymes were solved, and alternative substrates were tested. Crystal structures of the synthetases SfaD and SbnF were solved and compared to homologs from other species to define structural determinants of substrate preference. An analogue of SA, substituting D-lysine for D-ornithine during synthesis was produced in vitro and characterized using liquid chromatography and mass spectrometry. Furthermore, S. aureus was shown to be able to use this SA analogue for iron acquisition. Analogues of intermediates in the SB biosynthesis pathways were produced in vitro. The biosynthesis of a functional S. aureus siderophore analogue provided insights into the structures and substrate specificities of siderophore synthesis proteins. The modified siderophores may be of use to deliver antimicrobials into the cell or as a diagnostic for S. aureus infection.

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Initial Characterization of Peptidoglycan O-Acetylation and the Effects on Colonization Factors of Campylobacter Jejuni (2016)

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its pathogenesis is poorly understood. It lacks clear virulence factors such as those described for other enteropathogens. The characteristic helical shape of C. jejuni, maintained by the peptidoglycan (PG) layer, is important for colonization and host-pathogen interactions. Therefore, changes in morphology and the underlying PG greatly affect the physiology and biology of the organism. O-Acetylation of Peptidoglycan (OAP) is a phenomenon by which bacteria acetylate the C6 hydroxyl group of N-acetylmuramic acid in the glycan backbone to confer resistance to lysozyme and control lytic transglycosylase activity. The OAP gene cluster consists of a transmembrane PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a periplasmic PG O-acetyltransferase B (patB) responsible for O-acetylation of N-acetylmuramic acid (MurNAc), and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. Reduced OAP in ΔpatA and ΔpatB has a minimal effect on growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 results in marked differences in peptidoglycan biochemistry including changes in O-acetylation levels, anhydromuropeptide levels, and PG changes not expected to be a direct result of Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG metabolism. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibits defects in pathogenesis-associated phenotypes including cell shape, motility, biofilm formation, and sodium deoxycholate sensitivity. The mutant is also impaired for chick colonization and adhesion, and invasion and intracellular survival in INT407 epithelial cells lines in vitro. The importance of Ape1 activity to C. jejuni biology makes it a good candidate as a novel antimicrobial target.

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Structures of Photosynthetic Reaction Centers with Alternative Cofactors (2015)

Rhodobacter sphaeroides is a model organism for the study of bacterial photosynthesis. The R. sphaeroides photosynthetic reaction centre (RC) is the primary site of electron transfer, which is mediated by the photosynthetic pigments bacteriochlorophyll a (BChl) and bacteriopheophytin (BPhe). The substitution of key amino acid residues can change the type of cofactors present in the RC. In particular, studies have shown that when the leucine residue in position 214 of the M subunit [(M)L214] is converted into a histidine, the BPhe normally present in the neighbouring position (HA) is replaced with a BChl. This study investigated the hypothesis that steric exclusion by the coordinating residue causes dechelation of the central magnesium ion in BChl, producing BPhe. Crystal structures of RCs where (M)L214 is substituted for glycine and alanine were determined, which demonstrated that the presence of BPhe in the HA pocket is unchanged despite decreasing the size of the residue in position (M)214. A crystal structure of an RC where (M)L214 is substituted for asparagine was also determined and showed that the replacement of BPhe with BChl at HA occurs if residue (M)214 includes an amide moiety. In the R. sphaeroides Δbchd strain, which lacks the ability to make BChl, it is believed that the RC cofactor sites are populated exclusively with zinc-bacteriochlorophyll (Zn-BChl). The crystal structures of this Zn-BChl containing RC (Zn-RC) and a Zn-RC with the (M)L214H substitution (Zn-β-RC) were solved for the first time. These structures confirmed the presence of Zn-BChl in every cofactor position and the tetracoordination of the HA Zn-BChl in the Zn-β-RC, as well as revealing that the occupancy of the HB cofactor was much lower than that of all other cofactors.

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Structural basis for heme degradation in Staphylococcus aureus (2012)

IsdG and IsdI are heme degrading enzymes from the bacterium Staphylococcus aureus. Heme degrading enzymes use heme as a substrate and cofactor to degrade the porphyrin ring and release the central iron atom. Previous work on the structure of these enzymes showed metal coordination by a conserved His residue in the heme complex of the inactive IsdG-N7A variant and in the cobalt protoporphyrin IX complex of IsdI. In these structures, the porphyrin ring is highly distorted from planarity resulting in the β- and δ-meso carbons being displaced toward the distal side of the heme pocket and the α- and γ-meso carbons towards the His proximal side. This heme distortion is described as ruffling and is not present in the classical family of heme degrading enzymes called the heme oxygenases (HO) that bind heme in a planar manner. Thus, heme ruffling is proposed to be an important structural feature for the reaction mechanism of IsdG and IsdI. The role of heme ruffling in IsdI activity was examined in this study. For the first time, IsdI was cocrystallized in complex with its true substrate, heme, and the structure was solved to 1.50 Å resolution. The structure revealed extensive heme ruffling of 2.1 Å as determined by normal-coordinate analysis. IsdI was then engineered to adopt a flatter heme by mutating a conserved tryptophan residue, W66, in the heme pocket to the less bulky side chains of tyrosine, phenylalanine, leucine and alanine. Of the IsdI variants tested, only W66Y was amenable to X-ray structure solution. Heme ruffling in the variant was lessened to about 1.4 Å, demonstrating that W66 is an important contributor to heme distortion in IsdI. The activity of the W66Y variant was reduced to half that of wild-type suggesting a link between heme distortion and enzyme activity in IsdI.

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