Crosslinking of Dicyclotyrosine by the Cytochrome P450 Enzyme CYP121 from Mycobacterium tuberculosis Proceeds through a Catalytic Shunt Pathway [Molecular Bases of Disease]

CYP121, the P450 enzyme in Mycobacterium tuberculosis that catalyzes a single intramolecular C-C crosslinking 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. While 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 crosslinked 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 formation of a high-spin intermediate upon addition of peracetic acid to the enzyme-substrate complex. This intermediate exhibits a high-spin (S = 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, plausible free radical-based mechanisms for the C-C bond coupling reaction are proposed.

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