Unraveling the active site cover of coproheme decarboxylase from Listeria monocytogenes.

Coproheme decarboxylase (ChdC) is the terminal enzyme in Gram-positive heme b biosynthesis, an enzyme holding a special importance given its unique structure-function relationship and its necessity for bacterial survival. In the past, the enzyme has been shown to perform a double decarboxylation of two propionate groups on coproheme (Fe(III)-coproporphyrin III), subsequently converting it to heme b (iron-protoporphyrin IX). Notably, the active site of ChdCs is universally covered by a flexible loop. Its importance has not been fully studied but given its position it is assumed to provide steric hinderance for substrates or potential inhibitors to pass. This study aims to investigate its physiological role by introducing a histidine-to-alanine mutation located on the loop of ChdC from Listeria monocytogenes. Molecular dynamics simulation shows an increased flexibility of the structural element in the mutant. As a consequence, various kinetic studies at steady- and presteady-state conditions suggest coproheme binding and active site accessibility are improved. Using simulations and X-ray crystallography, we show evidence that the loop is originally stabilized by a hydrogen bond between S116 and the mutated H117. The higher accessibility also results in a higher susceptibility to damage from oxidative cosubstrates like H