Evolution and inhibition of cholesterol catabolising P450 enzymes in pathogenic mycobacteria
Date
2024
Authors
Doherty, Daniel Zocchi
Editors
Advisors
Bell, Stephen G.
Harris, Hugh
Harris, Hugh
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Thesis
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Abstract
Mycobacterium tuberculosis (Mtb), a globally distributed bacterium that has coexisted with humans for thousands of years, is a prominent example of a mycobacterial pathogen. Cholesterol catabolism by Mtb upon infection of a host is an important virulence factor, particularly during initial stages of infection, as it enables the bacteria to use cholesterol as a carbon source when enveloped by host macrophages. The targeting of this catabolic pathway has been suggested as a potential avenue for drug-discovery in an effort to overcome the rise in multi-drug resistant strains of tuberculosis (MDR-TB). Other pathogenic mycobacteria, including Mycobacterium ulcerans (Mulc), a bacterium that causes ulcerative skin lesion upon host infection, share homologous gene clusters with the cholesterol catabolism pathway of Mtb. This pathway is also shared by environmental bacteria and opportunistic pathogens such as Mycobacterium marinum (Mmar), a marine dwelling relative of both Mulc and Mtb. The conserved nature of this steroid catabolism pathway implicates its common ancestry in mycobacteria and is promising for the development of novel antibacterial therapies that may be effective for all of the aforementioned mycobacterial species. The P450 enzyme families CYP125 and CYP142 found within the Actinomycetota phylum (which includes mycobacteria) are responsible for the oxidation of a terminal methyl carbon of the cholesterol side-chain, which initiates side-chain degradation. CYP125 is the major enzyme family responsible for this process in vivo, though CYP142 can provide back-up functionality in some species. CYP142 has also been proposed as a more efficient cholesterol ester side-chain oxidising enzyme than CYP125. The side-chain degradation releases two units of propionyl coenzyme A (propionyl- CoA) and one of acetyl coenzyme A (acetyl-CoA), which provide mycobacteria with both a carbon source for central metabolism and components required to biosynthesise surface virulence lipids that are associated with pathogenicity. A greater understanding of the respective functions of CYP125 and CYP142, including their differences between mycobacteria, would aid in the process of targeted drug-development efforts. Furthermore, an investigation of the evolution of the CYP125 family would uncover any changes at the amino-acid level that may have enhanced the pathogenicity of certain mycobacteria like Mtb. The work presented in this thesis provides insight into the structure, function and evolution of the cholesterol side-chain oxidising P450 enzyme families CYP125 and CYP142 present in mycobacteria. Firstly, the crystal structures of cholest-4-en-3-one-bound CYP142A3 enzymes from Mmar and Mulc were determined, as well as their activity to a range of steroid substrates including cholesterol and cholest-4-en-3-one. Not only was it found that the structures of these two enzymes were highly conserved with each other, but also with CYP1421 from Mtb. Cholest-4-en-3-one also maintained an almost identical binding mode across the three enzyme-substrate structures. Activity assays conducted in vitro demonstrated that both CYP142A3 enzymes and CYP142A1 were able to bind and oxidise cholesterol, cholest-4-en-3-one and cholesteryl sulfate, with each producing the 26-hydroxylated and 26-carboxylated metabolites. Similar assays using a combination of 98% deuterated cholesterol and cholesterol revealed no preference for either substrate, indicating that initial C-H bond abstraction is not rate limiting in CYP142- mediated steroid oxidation. Secondly, phytosterol (plant sterol) oxidation within the CYP125 enzyme family was established in vitro, a function that was previously assumed but not experimentally demonstrated. All three of MulcCYP125A7, MmarCYP125A6 and MtbCYP125A1 were able to oxidise sitosterol to 26-hydroxysitosterol, with MmarCYP125A6 having the additional capacity to oxidise this intermediate further to 26-sitostenoic acid. Similarly, oxidation of campesterol was observed for all three of these CYP125 enzymes. Phytosterol oxidation was successful despite the lack of significant UV-Vis absorbance spectral perturbations of the P450 heme Soret band, a feature that is usually observed for P450 substrates. In contrast, the activity of CYP142A1 and CYP124A1 for sitosterol was significantly lower, a finding which supports the notion that CYP125 functionality is not completely backed-up by CYP142 or CYP124. Competitive oxidation assays of a 1:1 mixture of sitosterol and cholesterol showed that MtbCYP125A1 had a slightly higher preference for cholesterol relative to the other CYP125 enzymes tested, suggesting that the former enzyme may have evolved to be more efficient for human sterol oxidation upon host infection. Thirdly, a set of cholesterol-based Mtb inhibitors, synthesised by collaborators at the University of Queensland, were screened for binding to MulcCYP125A7, Mulc- CYP142A3 and MtbCYP125A1, adding to previously collected data for the Mmar equivalents of these enzymes. These were based on the 16β,26-dihydroxycholesterol, a triol that had previously been shown to have antibacterial potency against Mtb and to have bound to MtbCYP125A1. Initial findings demonstrated that compounds that possessed polar functionalities at C-3, C-16 and C-26 in the cholesterol scaffold induced Reverse Type I (RTI) UV-Vis spectral shifts in the heme Soret band, implying indirect binding of the inhibitor to the heme. Removal of the C-16 hydroxyl moiety and the introduction of a triazole group at C-26 produced the compound I22, which demonstrated the first conclusive evidence from these sets of inhibitors of a close enough epproach to enable the direct coordination of the ligand to the heme centre. I22 bound to CYP125A1 with a Kd of 1.13 μM, and produced antimicrobial activity with a MIC90 of 1.3 μM. Further screening demonstrated that I22 directly coordinated to the heme of all CYP125 and CYP142 enzymes from Mmar, Mulc and Mtb, and also Mmar- CYP124A1. These results provide a promising starting point for the development of a more drug-like inhibitor based on the structure of I22, that can inhibit all cholesterol side-chain oxidising P450 enzymes in mycobacteria. Finally, insight into the evolutionary history of the CYP125 enzyme family was provided by the construction of a maximum-likelihood, most-recent common ancestor, CYP125MRCA, using Ancestral Sequence Reconstruction (ASR). Reconstruction of the hypothesised CYP125MRCA protein using ASR, followed by protein production in E. coli and purification allowed for comparison to the extant CYP125 enzymes previously discussed. The melting temperature (Tm) of CYP125MRCA was 64.4 ± 0.8 °C, approximately 10 degrees higher than MulcCYP125A7, evidencing the higher thermostability of the ancestral enzyme. Oxidation assays revealed that CYP125MRCA was able to bind and oxidise the same phytosterols and animal sterols as its extant relatives. Binding of phytosterols to CYP125MRCA, unlike previously tested extant enzymes, shifted the Soret band of the protein’s UV-Vis spectrum in a typical Type I fashion, implying that these substrates were more capable of displacing the heme’s axial water ligand in the ancestral enzyme. In contrast to CYP125A1, CYP125MRCA demonstrated a clear preference for phytosterols, consistent with an evolutionary history as an environmental organism rather than an obligate pathogen. Vitamin D3 was also successfully bound and oxidised by CYP125MRCA but not by MulcCYP125A7 or CYP125A1, evidencing an increased substrate range in the ancestor. The X-ray crystal structure of CYP125MRCA in complex with vitamin D3 established its binding mode, and comparison with that of cholest-4-en-3-one-bound CYP125A1 highlighted an active-site pocket in the ancestor, one that was not present in CYP125A1, that enabled the binding of vitamin D3. Both C-25 and C-26 hydroxylation of vitamin D3 was observed. The production of 25-hydroxyvitamin D3, a pharmaceutically important compound, demonstrated the potential utility of ASR for the generation of valuable compounds.
School/Discipline
School of Physics, Chemistry and Earth Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Physics, Chemistry and Earth Sciences, 2024
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