Cross-linking of dicyclotyrosine by the cytochrome P450 enzyme CYP121 from Mycobacterium tuberculosis proceeds through a catalytic shunt pathway

Abstract

CYP121, the cytochrome P450 enzyme in that catalyzes a single intramolecular C-C cross-linking reaction in the biosynthesis of mycocyclosin, is crucial for the viability of this pathogen. This C-C coupling reaction represents an expansion of the activities carried out by P450 enzymes distinct from oxygen insertion. Although the traditional mechanism for P450 enzymes has been well studied, it is unclear whether CYP121 follows the general P450 mechanism or uses a different catalytic strategy for generating an iron-bound oxidant. To gain mechanistic insight into the CYP121-catalyzed reaction, we tested the peroxide shunt pathway by using rapid kinetic techniques to monitor the enzyme activity with its substrate dicyclotyrosine (cYY) and observed the formation of the cross-linked product mycocyclosin by LC-MS. In stopped-flow experiments, we observed that cYY binding to CYP121 proceeds in a two-step process, and EPR spectroscopy indicates that the binding induces active site reorganization and uniformity. Using rapid freeze-quenching EPR, we observed the formation of a high-spin intermediate upon the addition of peracetic acid to the enzyme-substrate complex. This intermediate exhibits a high-spin ( = 5/2) signal with g values of 2.00, 5.77, and 6.87. Likewise, iodosylbenzene could also produce mycocyclosin, implicating compound I as the initial oxidizing species. Moreover, we also demonstrated that CYP121 performs a standard peroxidase type of reaction by observing substrate-based radicals. On the basis of these results, we propose plausible free radical-based mechanisms for the C-C bond coupling reaction.

Probing Ligand Exchange in the P450 Enzyme CYP121 from Mycobacterium tuberculosis: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature

“…In our recent study, we found that peracetic acid can oxidize the CYP121-cYY complex via a peroxide shunt mechanism, in which we observed the rapid formation of a probable ferric alkyl-peroxo species. 15 In the later steps of the reactions it catalyzes, CYP121 functions more like a peroxidase than a typical P450. 15 We noticed that, from the very first reaction step, CYP121 does not follow the general P450 chemical mechanism.…”

“…15 In the later steps of the reactions it catalyzes, CYP121 functions more like a peroxidase than a typical P450. 15 We noticed that, from the very first reaction step, CYP121 does not follow the general P450 chemical mechanism. In the generally accepted mechanism of P450 enzymes, binding of the primary organic substrate results in release of the axial water ligand.…”

“…16 The UV–vis data published suggest a spin transition due to binding of the substrate, but the previous EPR studies do not show the low-spin to high-spin transition. 14,20,15 Here, we apply structural, spectroscopic, and kinetic experiments to investigate the equilibria among various heme states as a function of pH and temperature. We also investigated the rate of metal-ligand exchange using the diatomic molecule, NaCN.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…287 Given the essential nature of CYP121 to the survival of M. tuberculosis, 288 this P450 has been extensively characterised by a number of groups, which have afforded structural, biochemical and mechanistic insights into the function of this P450. [289][290][291] Mechanistically, the formation of the C-C bond (similar to aviolin) has been postulated to occur either by via proton coupled electron transfer or electron tunnelling between the two aromatic rings, 177,292 which is a needed to avoid having to completely re-orient the substrate within the active site during catalysis, which was an early mechanistic postulate in this case. 287 One of the more impressive series of transformations reported to occur during bacterial DKP synthesis is found in the biosynthesis of bicyclomycin.…”

“…From a mechanistic perspective, the crosslinking reaction likely occurs by hydrogen abstraction from the ring closest to the heme iron, with subsequent generation of a radical species in the distal aviolin occurring either via proton coupled electron transfer or electron tunnelling as has been postulated for the crosslinking reaction performed by CYP121 (vide infra). 177 A further interesting note in the investigation of CYP158 enzymes has been their use in mechanistic studies, where the Green group has been able to use these enzymes to investigate the highly reactive intermediates that are found within the P450 active cycle. …”