The structure ofMycobacterium tuberculosisheme-degrading protein, MhuD, in complex with product

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, both in sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with heme substrate, no structures have been reported of IsdG-type proteins in complex with product, unlike HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXa (aBV) rather than the wild-type (WT) mycobilin isomers as product. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product aBV as a proxy to study the IsdG-type protein/product complex. First we show that aBV has nanomolar affinity for MhuD and the R26S variant. Second we determined the MhuD-R26S-aBV complex structure to 2.5 Å, which reveals two notable features (1) two aBV molecules bound per active site and (2) a new a-helix (a3) as compared with the MhuD-heme structure. Finally, by molecular dynamics simulations we show that a3 is stable with the proximal aBV alone. MhuD's high affinity for its product and structural and electrostatic changes that accompany substrate turnover suggest that there is an unidentified protein that is responsible for product extraction from MhuD and other IsdG-type proteins.