Methyltransferase CPmtA Drives Dormancy-Associated Synthesis of Coproporphyrin III Tetramethyl Ether Via a “carousel” Mechanism in Mycobacterium Smegmatis

It was shown previously that mycobacteria (Mycobacterium smegmatis and Mycobacterium tuberculosis) accumulate tetramethyl ether of coproporphyrin III (TMC) during transition to dormant state. However, the enzymes involved in TMC synthesis remained unknown. Here we have identified a new M. smegmatis methyltransferase MSMEG_0614 (CPmtA) that catalyzes the S-adenosyl-L-methionine-dependent conversion of coproporphyrin (CP) into TMC through sequential methylation of its carboxyl groups. Overexpression of MSMEG_0614 in both M. smegmatis and M. tuberculosis leads to increase in intracellular TMC levels and reduction of bacterial respiratory activity, linking this enzymatic pathway to dormant state transition in mycobacteria. Structural-dynamic modeling reveals a unique “carousel” mechanism in which the orientation of CP alternates within the enzyme's active site to methylate its carboxyl groups sequentially. Mutagenesis based on computational predictions validates the catalytic mechanism. In addition, simulations demonstrate that TMC, unlike CP, integrates into a model phospholipid membrane, altering its properties, which possibly are important for transition to dormancy.