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Investigating a novel protein-based cofactor: toward elucidating the catalase mechanism of Mycobacterium tuberculosis KatG


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dc.contributor.advisorGoodwin, Douglas
dc.contributor.authorAziz, Tarfi
dc.date.accessioned2022-12-07T21:00:18Z
dc.date.available2022-12-07T21:00:18Z
dc.date.issued2022-12-07
dc.identifier.urihttps://etd.auburn.edu//handle/10415/8536
dc.description.abstractCatalase-peroxidases (KatGs) have been engineered by nature to exhibit dual functionalism, degrading H2O2 by catalase and peroxidase mechanisms. This serves to protect the organisms that carry it (primarily bacteria and fungi) against peroxide-dependent oxidative damage. Despite bearing no resemblance to monofunctional (i.e., typical) catalases, KatGs have robust catalase activity due at least in part to a novel covalent linkage between three side chains (by Mycobacterium tuberculosis KatG [Mtb KatG] numbering, Met 255, Tyr 229, and Trp 107) (MYW). This MYW cofactor redox cycles between its radical (MYW•+) and fully covalent states, enabling KatG to leverage heme intermediates for catalatic O2 production. However, the molecular mechanism by which the adduct is formed and how this unique structure contributes to overall catalytic mechanism of KatG have yet to be fully elucidated. Here, site-directed mutagenesis in combination with optical stopped-flow spectrophotometry and rapid freeze-quench EPR spectroscopy have been used to investigate the mechanism of MYW adduct formation and the catalase activity that emerges from it. In order to pursue these lines of research, it was essential to develop a robust and reliable protocol for producing KatG which possessed its heme prosthetic group but had not yet engaged in the autocatalytic formation of its MYW cofactor. Historically, it has always been challenging to produce KatG that contains heme, but at the same time, lacks this critical post-translational modification, and is capable of forming the adduct on demand and displaying the full range of KatG’s catalytic abilities. Due to the large size of the protein (80 kDa), denaturation or unfolding has never been a successful strategy to regain the protein in the native form. Moreover, use of harsh organic compounds for denaturing has also made the protein vulnerable for mechanistic investigation. Throughout the field of KatG biochemistry, heme is incorporated during expression. Exposure to endogenous peroxides or other oxidants during expression ensures that the purified enzyme product invariably contains the fully formed MYW cofactor, making it impossible to evaluate the steps of its formation. The alternative is to produce KatG such that heme is not incorporated. That is, that apoKatG (aKatG) would be expressed and purified. The aKatG must then be successfully reconstituted with the heme cofactor after isolation from the expression culture to prevent premature peroxide-induced MYW formation. This challenge has thwarted the KatG field for many years. As described in Chapter 2 of this dissertation, we were able to develop a protocol which solved this problem. Using a standard E. coli laboratory expression strain (BL-21 [DE3] pLysS) and withholding heme and heme precursors, we were able to express KatG from which the overwhelming product was the aKatG form. We were able to reconstitute aKatG with heme in the midst of the purification process to generate the reconstituted form (rKatG). Structural consistency and fully functional capacity of rKatG were demonstrated by strong and typical UV absorption features and enzymatic activity. The striking yield of rKatG (at least 20-fold higher) is one of the most fortuitous aspects of this study and created the possibility of using more sophisticated and material-intensive techniques like rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) spectroscopy and Mössbauer spectroscopy. By all measures employed to date, rKatG is indeed a suitable tool for mechanistic investigation. With separate protocols in hand to generate reconstituted and mature forms of WT KatG (rWT and mWT KatG, respectively), we evaluated and compared each form for their reactions with peroxides using optical stopped-flow and RFQ-EPR spectroscopy (Chapter 3). Under multiple-turnover conditions using H2O2, optical stopped-flow experiments showed an initial appearance of a high-valent ferryl-like (FeIV=O) intermediate instead of the intermediate typically observed for KatG engaging in catalatic H2O2 decomposition under steady-state conditions, a FeIII-O2•--like species. Nevertheless, in contrast to catalase-negative canonical heme peroxidases, full catalase H2O2 decomposition did emerge over the course of the reaction. This was evident in two ways. First, the resting or FeIII form of the enzyme reemerged at the conclusion of the reaction, indicating that the balance of H2O2 present at the start of the reaction had been depleted. Second, the initial FeIV=O state observed early in the reaction gave way to the FeIII-O2•--like KatG catalase steady-state species. This latter transition was accompanied by an irreversible increase in absorbance near 315 nm, consistent with the establishment of the MYW cofactor (or precursors thereof). Interestingly, pretreatment of rWT KatG with limited molar equivalents of peracetic acid (PAA), a peroxide that supports only heme/enzyme oxidation, produced an enzyme with the properties of the mKatG enzyme. EPR experiments revealed that an admixture of radical species appeared before any evidence of a MYW cofactor radical intermediate, suggestive of the preferred site of crosslink initiation. At the earliest reaction time (6 ms), rKatG produced an exchange-broadened radical signal followed by a narrow doublet signal. Such an early appearance of an exchange-coupled radical is not observed with mKatG when it is reacted with its natural substrate, H2O¬2. Rather, this type of signal is only detected upon reaction with peroxides incapable of supporting catalatic turnover (i.e., peracetic acid). Reactions of mKatG with H2O2 invariably produce the narrow doublet radical assigned as the MYW•+ species. Thus, we propose that such an exchange-broadened species arises from a radical centered on the distal Trp prior to its incorporation into the MYW cofactor. Variants of KatG incapable of establishing the MYW adduct (M255I, Y229F and W107F) were also constructed, expressed, purified, and reconstituted with heme. Stopped-flow and UV-vis analyses of these variants showed disrupted catalase activities and incomplete catalatic turnover. Interestingly, partial adduct bearing rM255I supported catalatic turnover at a rate greater than other adduct negative variants at low pH (i.e., pH 5.0). As compared to the WT activity, the rate and extent of O2 was relatively low; however, measurable O2 production above that of typical peroxidases has not been reported among these adduct negative variants. These observations strongly suggest that the distal side Trp 107 and Tyr 229 are essential components of the catalase mechanism; limited catalase activity can be accomplished with just those two residues, but the full activity of the enzyme cannot be achieved without the sulfonium linkage provided by Met 255. Our reconstituted KatG proteins may permit investigation of radical transfer reactions leading to formation of KatG's novel MYW cofactor as well as the influence of other protein radical transfer reactions on that process. However, as far as the molecular mechanism is concerned, a main limitation of steady state kinetics is that the nature of the rate-limiting step in the global catalytic process cannot always be established with certainty, so that it is not always clear which step(s) the limits the overall rate. We propose that a technique combining rapid quench with mass spectrometry for detecting and quantitating the covalent intermediates and products of MYW cofactor formation. Used in combination with the kinetics of adduct formation monitored at 315 nm and the transient kinetics of radical species observed by EPR, a thorough elucidation of the mechanism of MYW adduct formation is anticipated. Another important aspect of this study was the evaluation of protein-based radicals alongside the kinetics of formation and decay of transient heme states. To a significant degree continuous-wave, X-band EPR and optical absorption measurements, respectively, carry with them ambiguity that is addressed by alternative and complementary techniques. Signals that we obtained from conventional EPR measurement creates ambiguity due to resemblance in shape and relaxation behavior of the Tyr or Trp radical. Further confirmation requires isotopic labeling of tyrosine and tryptophan residues, nitric oxide radical trapping techniques, and/or 57Fe-labeling to verify the influence of the heme iron on protein-based radicals in very close proximity to the active site. The latter is also essential for Mössbauer studies which provide more definitive assignment of iron oxidation states, particularly those that are EPR silent. Our protocol for expressing KatG without heme followed by successful reconstitution with the cofactor after purification not only enabled the studies described in this dissertation, but also make possible these more advanced and material-intensive spectroscopic strategies. A complete understanding of radical transfer processes is the key to unlocking all aspects of KatG catalysis, including but not limited to MYW cofactor generation and utilization, through-protein radical transfer as an enzyme activity fail-safe. These are the central to KatG’s role in important physiological processes like host-pathogen interactions and isoniazid activation. This work was supported by a grant from the National Science Foundation (MCB 1616059)en_US
dc.rightsEMBARGO_NOT_AUBURNen_US
dc.subjectChemistry and Biochemistryen_US
dc.titleInvestigating a novel protein-based cofactor: toward elucidating the catalase mechanism of Mycobacterium tuberculosis KatGen_US
dc.typePhD Dissertationen_US
dc.embargo.lengthMONTHS_WITHHELD:12en_US
dc.embargo.statusEMBARGOEDen_US
dc.embargo.enddate2023-12-07en_US
dc.contributor.committeeDuin, Eduardus
dc.contributor.committeeCalderon, Angela
dc.contributor.committeeEllis, Holly

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