Kirsty J Mclean

Kirsty J Mclean
University of Huddersfield · Department of Chemical and Biological Sciences

Doctor of Philosophy

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114
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Publications

Publications (114)
Article
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There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a vali...
Article
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CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small mo...
Article
A series of analogues of cyclo(L-tyrosyl-L-tyrosine), the substrate of the Mycobacterium tuberculosis enzyme CYP121, have been synthesized and analyzed by UV-Vis and EPR spectroscopy and by X-ray crystallography. The introduction of iodine substituents onto cyclo(L-tyrosyl-L-tyrosine) results in sub-μM binding affinity for the CYP121 enzyme and a c...
Article
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The emergence of untreatable drug‐resistant strains of Mycobacterium tuberculosis is a major public health problem worldwide, and the identification of new efficient treatments is urgently needed. Mycobacterium tuberculosis cytochrome P450 CYP121A1 is a promising drug target for the treatment of tuberculosis owing to its essential role in mycobacte...
Article
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Flavocytochrome P450 BM3 is a natural fusion protein constructed of cytochrome P450 and NADPH-cytochrome P450 reductase domains. P450 BM3 binds and oxidizes several mid- to long-chain fatty acids, typically hydroxylating these lipids at the ω-1, ω-2 and ω-3 positions. However, protein engineering has led to variants of this enzyme that are able to...
Article
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The rise in multidrug resistant (MDR) cases of tuberculosis (TB) has led to the need for the development of TB drugs with different mechanisms of action. The genome sequence of Mycobacterium tuberculosis (Mtb) revealed twenty different genes coding for cytochrome P450s. CYP121A1 catalyzes a C–C crosslinking reaction of dicyclotyrosine (cYY) produci...
Article
The CYP152 family of cytochrome P450 enzymes (P450s or CYPs) are bacterial peroxygenases that use hydrogen peroxide to drive hydroxylation and decarboxylation of fatty acid substrates. We have expressed and purified a novel CYP152 family member - CYP152K6 from the methylotroph Bacillus methanolicus MGA3. CYP152K6 was characterized using spectroscop...
Article
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Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio- and stereoselective CH-activating oxidation of structurally simple and complex organic compounds su...
Article
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The cytochromes P450 (P450s or CYPs) constitute a large heme enzyme superfamily, members of which catalyze the oxidative transformation of a wide range of organic substrates, and whose functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The P450 peroxygenases are a subgroup of the P450s that have...
Chapter
The cytochrome P450 monooxygenase enzymes (P450s) catalyze a diverse array of chemical transformations, most originating from the insertion of an oxygen atom into a substrate that binds close to the P450 heme. The oxygen is delivered by a highly reactive heme iron-oxo species (compound I) and, according to the chemical nature of the substrate and i...
Article
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Three series of biarylpyrazole imidazole and triazoles are described, which vary in the linker between the biaryl pyrazole and imidazole/triazole group. The imidazole and triazole series with the short –CH2- linker displayed promising antimycobacterial activity, with the imidazole–CH2- series (7) showing low MIC values (6.25-25 μg/mL), which was al...
Article
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Cytochrome P450 BM3 is an important model system for mammalian P450s and has great potential for biotechnological applications owing to its high catalytic activity, facilitated by its fused diflavin reductase domain. Mutagenesis studies have shown that Ala-82 and Phe-87 are important in controlling molecular selectivity and regioselectivity in BM3...
Article
Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, serie...
Article
Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-in...
Article
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The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridec...
Article
Native mass spectrometry is an ideal technique to investigate the effects of DMSO on protein structure and interac-tions. Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on...
Article
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Similarity in the ligand binding profile of two enzymes may provide insight for functional characterization and be of greater relevance to inhibitor development than sequence similarity or structural homology. Fragment screening is an efficient approach to characterizing the ligand profile of an enzyme and has been applied here to study the family...
Article
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Jeotgalicoccus sp. 8456 OleTJE (CYP152L1) is a fatty acid decarboxylase cytochrome P450 that uses hydrogen peroxide (H2 O2 ) to catalyse production of terminal alkenes, which are industrially important chemicals with biofuel applications. We report enzyme fusion systems in which Streptomyces coelicolor alditol oxidase (AldO) is linked to OleTJE . A...
Article
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The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representat...
Article
Full-text available
The Jeotgalicoccus sp. peroxygenase cytochrome P450 OleTJE (CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleTJE could enable pro...
Article
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The Mycobacterium tuberculosis (Mtb) H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many Mtb P450s remain uncharacterized, suggesting their further analysis may provide new insights into Mtb metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family wi...
Article
TB, caused by the human pathogen Mycobacterium tuberculosis (Mtb, causes more deaths than any other infectious disease. Iron is crucial for Mtb to infect the host and to sustain infection, with Mtb encoding large numbers of iron-binding proteins. Many of these are hemoproteins with key roles, including defense against oxidative stress, cellular sig...
Article
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The cyclo-dipeptide substrates of the essential M. tuberculosis (Mtb) enzyme CYP121 were deconstructed into their component fragments and screened against the enzyme. A number of hits were identified, one of which exhibited an unexpected inhibitor-like binding mode. The inhibitory pharmacophore was elucidated, and fragment binding affinity was rapi...
Article
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Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in specie...
Article
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The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound...
Article
Cytochrome P450 enzymes (P450s or CYPs) catalyze an enormous variety of oxidative reactions in organisms from all major domains of life. Their monooxygenase activity relies on the reductive scission of molecular oxygen (O2) bound to P450 heme iron, and thus on the delivery of two electrons to the heme iron at discrete points in the catalytic cycle....
Article
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The microbial P450s perform an array of oxidative and other chemical reactions that are both crucial for the viability of the bacterial, archaeal, and fungal hosts, and which have numerous important applications. The soluble nature of the bacterial and archaeal P450s has facilitated their expression and purification in high yields, and has enabled...
Article
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The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway in...
Article
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Some bacteria and archaea synthesise haem by an alternative pathway, which involves the sequestration of sirohaem as a metabolic intermediate rather than as a prosthetic group. Along this pathway the two acetic acid side chains attached to C12 and C18 are decarboxylated by sirohaem decarboxylase, a heterodimeric enzyme composed of AhbA and AhbB, to...
Article
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Production of drug metabolites is one area where enzymatic conversion has significant advantages over synthetic chemistry. These high value products are complex to synthesise, but increasingly important in drug safety testing. The vast majority of drugs are metabolized by cytochrome P450s (P450s), with oxidative transformations usually being highly...
Article
We present a novel fragment-based approach that tackles some of the challenges for chemical biology of predicting protein function. The general approach, which we have termed biofragments, comprises two key stages. First, a biologically relevant fragment library (biofragment library) can be designed and constructed from known sets of substrate-like...
Article
The heme-containing cytochrome P450s exhibit isoform-dependent ferric spin equilibria in the resting state and differential substrate-dependent spin equilibria. The basis for these differences is not well understood. Here, magnetic circular dichroism (MCD) reveals significant differences in the resting low spin ligand field of CYPs 3A4, 2E1, 2C9, 1...
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The production of hydrocarbons in nature has been documented for only a limited set of organisms, with many of the molecular components underpinning these processes only recently identified. There is an obvious scope for application of these catalysts and engineered variants thereof in the future production of biofuels. Here we present biochemical...
Article
The truncated hemoglobin from Bacillus subtilis (trHb-Bs) possesses a surprisingly high affinity for oxygen and resistance to (auto)oxidation; its physiological role in the bacterium is not understood and may be connected with its very special redox and ligand binding reactions. Electron transfer reactions of trHb-Bs were electrochemically studied...
Article
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Freedom to merge: A combination of crystal structure examination and in silico predictions made it possible to overcome the conformational limitations of fragment merging and escape the internal strain in a series of weakly binding merged fragments that target M. tuberculosis CYP121. The insights attained provide a new perspective and guide for pri...
Article
Nanoelectrospray ionisation mass spectrometry (nanoESI MS) of intact protein complexes was used to study CYP121, one of the twenty cytochrome P450s in Mycobacterium tuberculosis (Mtb) and an enzyme that is essential for bacterial viability. The results shed new light on both ligand-free and ligand-bound states of CYP121. Isolated unbound CYP121 is...
Article
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Despite the extensive study of the biosynthesis of the complex molecule B (cobalamin), the mechanism by which the lower ligand 5,6-dimethylbenzimidazole (DMB) is formed has remained something of a mystery. However, recent work has identified and characterized a DMB-synthase (BluB) responsible for the oxygen-dependent, single enzyme conversion of FM...
Data
Rates of peroxyflavin formation and degradation; variable BluB concentration. The figure shows the apparent rate of the formation of c4a-peroxyflavin in the presence of a variable concentration of (RC)BluB (black circles). The concentration of FMNH2 remained constant at 171 µM and the absorbance was measured at 380 nm. The degradation of the peroxy...
Data
Subtraction spectrum. Dashed line shows the initial spectrum of BluB and FMNH2 combined with oxygenated buffer (291 µM FMNH2 was incubated with 684 µM (RC)BluB and prior to stopped flow mixing with oxygenated buffer A. Spectrum recorded 2.5 s after mixing), dotted line shows the initial spectrum of the control sample (291 µM FMNH2 combined in the s...
Data
Analysis of the flavin cofactor bound to CbiY. HPLC chromatogram and ESI +ve mass spectrum of the flavin cofactor isolated from CbiY. Retention time and MS were consistent with the bound cofactor being FMN (m/z 457 [M+H]+). (DOCX)
Data
Control stopped flow experiment. FMNH2 (291 µM) combined in the stopped flow with oxygenated buffer A. Spectra were collected every 2.5 s. An increase in absorbance at 375 nm and 460 nm (red) indicate a reoxidation of free FMN. This was followed by a loss of absorbance (black) over time probably due to photobleaching of the flavin. (DOC)
Article
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The cytochromes P450 (P450s) are probably nature's most versatile enzymes in terms of both their vast substrate range and the diverse types of molecular transformations performed across the P450 enzyme superfamily. The P450s exquisitely perform highly specific oxidative chemistry, utilizing a sophisticated catalytic reaction mechanism. Recent studi...
Chapter
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SynonymsRedox cofactorsDefinitionElectron transfer cofactors are non-protein molecules that bind to proteins and enzymes and act as conduits for the passage of electrons in redox reactions.IntroductionLife in all organisms relies on electron transport to facilitate molecular transformations and for fundamental processes such as respiration and phot...
Article
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Pieces of the puzzle: The first fragment-based approach was used to target cytochrome P450 enzymes (CYPs) for drug development. The experiments provide new insights into the binding site of the essential Mycobacterium tuberculosis CYP121 enzyme, and resulted in a promising novel lead compound based on fragment merging.
Article
Teile des Puzzles: Ein fragmentbasierter Ansatz wurde erstmals zur Untersuchung von Cytochrom‐P450‐Enzymen (CYPs) in der Wirkstoffentwicklung eingesetzt (siehe Schema). Die Experimente bieten neue Einblicke in die Bindungsstellen des CYP121‐Enzyms aus Mycobacterium tuberculosis und führten zu einer vielversprechenden neuen Leitverbindung.
Article
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TB (tuberculosis) disease remains responsible for the death of over 1.5 million people each year. The alarming emergence of drug-resistant TB has sparked a critical need for new front-line TB drugs with a novel mode of action. In the present paper, we review recent genomic and biochemical evidence implicating Mycobacterium tuberculosis CYP (cytochr...
Article
Full-text available
Cholesterol is an essential molecule for eukaryotic life and is an important precursor for a wide range of physiological processes. Biosynthesis and homoeostasis of cholesterol are complex mechanisms that are tightly regulated and interlinked with activities of a number of cytochrome P450 enzymes. These P450s play central critical roles in choleste...
Article
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The Rhodococcus rhodochrous strain 11Y XplA enzyme is an unusual cytochrome P450-flavodoxin fusion enzyme that catalyzes reductive denitration of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX). We show by light scattering that XplA is a monomeric enzyme. XplA has high affinity for imidazole (K(d) = 1.6 μM), explaining previous reports...
Article
The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and a...
Article
Wheat leaves contain two isoproteins of the photosynthetic ferredoxin:NADP(+) reductase (pFNRI and pFNRII). Truncated forms of both enzymes have been detected in vivo, but only pFNRII displays N-terminal length-dependent changes in activity. To investigate the impact of N-terminal truncation on interaction with ferredoxin (Fd), recombinant pFNRII p...
Article
Cytochrome P450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)═O]+• termed Compound I (Cpd I). The proximal cysteine thiolate ligand is a critical determinant of the spectral and catalytic properties of P450 enzymes. To explore the effect of an increased level of donation of electr...
Article
CYP144 from Mycobacterium tuberculosis was expressed and purified. CYP144 demonstrates heme thiolate coordination in its ferric form, but the cysteinate is protonated to thiol in both the carbon monoxide-bound and ligand-free ferrous forms (forming P420 in the former). Tight binding of various azole drugs was shown, with affinity for miconazole (K(...