Ferrochelatase HemH Research Articles

Staphylococcus aureus haem biosynthesis: characterisation of the enzymes involved in final steps of the pathway.

Haem is a life supporting molecule that is ubiquitous in all major kingdoms. In Staphylococcus aureus, the importance of haem is highlighted by the presence of systems both for the exogenous acquisition and endogenous synthesis of this prosthetic group. In this work, we show that in S. aureus the formation of haem involves the conversion of coproporphyrinogen III into coproporphyrin III by coproporphyrin synthase HemY, insertion of iron into coproporphyrin III via ferrochelatase HemH, and oxidative decarboxylation of Fe-coproporphyrin III into protohaem IX by Fe-coproporphyrin oxidase/dehydrogenase HemQ. Together, this route represents a transitional pathway between the classic pathway and the more recently acknowledged alternative biosynthesis machinery. The role of the haem biosynthetic pathway in the survival of the bacterium was investigated by testing for inhibitors of HemY. Analogues of acifluorfen are shown to inhibit the flavin-containing HemY, highlighting this protein as a suitable target for the development of drugs against S. aureus. Moreover, the presence of a transitional pathway for haem biosynthesis within many Gram positive pathogenic bacteria suggests that this route has the potential not only for the design of antimicrobials but also for the selective discrimination between bacteria operating different routes to the biosynthesis of haem.

Probing the structural plasticity of an archaeal primordial cobaltochelatase CbiXS

Insertion of metal ions into tetrapyrrole macrocycles is catalyzed by a diverse group of enzymes called chelatases. Structures are known for several chelatases catalyzing metal insertion into protoporphyrin IX or sirohydrochlorin. Despite a lack of significant amino acid sequence similarity, these ferro- and cobaltochelatases share a high degree of structural similarity. Cobaltochelatase CbiK and ferrochelatase HemH are bilobial enzymes with two alpha/beta domains, which were suggested to origin from a common ancestral protein via gene duplication. Small, single-domain chelatases (CbiX(S)) were recently described in archaea and are believed to represent primordial chelatases. Here, we tested the structural plasticity of an archaeal cobaltochelatase CbiX(S) by rearranging its structure with a novel method producing random in-frame deletions, duplications and insertions. A number of functional chelatase variants with insertion of duplicated sequence stretches, encompassing from one to nine secondary structural elements, were obtained. CbiX(S) was found to tolerate large sequence rearrangements in four out of the nine loop regions of the protein, indicating a high degree of structural plasticity. The predicted topologies of two variants (M51 and M518) are strikingly similar to CbiK and HemH, suggesting that we recreated duplication events that are believed to have created the bilobial chelatases.