Structure of a Mycobacterium tuberculosis Heme-Degrading Protein, MhuD, Variant in Complex with Its 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, in both 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 the heme substrate, no structures of IsdG-type proteins in complex with a product have been reported, unlike the case for HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXα (αBV) rather than the wild-type mycobilin isomers. 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 αBV as a proxy to study the IsdG-type protein–product complex. First, we show that αBV has a nanomolar affinity for MhuD and the R26S variant. Second, we determined the MhuD-R26S−αBV complex structure to 2.5 Å, which reveals two notable features: (1) two αBV molecules bound per active site and (2) a novel α-helix (α3) that was not observed in previous MhuD–heme structures. Finally, through molecular dynamics simulations, we show that α3 is stable with the proximal αBV alone. MhuD’s high affinity for the product and the observed structural and electrostatic changes that accompany substrate turnover suggest that there may be an unidentified class of proteins that are responsible for the extraction of products from MhuD and other IsdG-type proteins.

Biosynthesis of the modified tetrapyrroles—the pigments of life

“…Staphylococcus aureus produces two members of the IsdG family, IsdG and IsdI, which produce formaldehyde and "staphylobilin," a mixture of 5-oxo-d-bilirubin and 5-oxo-b-bilirubin (358). The MhuD enzyme of Mycobacterium tuberculosis produces mycobilin, which is a derivative of 5-oxob-bilirubin in which the bridging carbon is retained as a formyl group (359)(360)(361). The model green alga, Chlamydomonas reinhardtii, can produce two members of the HO-1 family and one member of the IsdG family.…”

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

“…A likely source of the discrepancy between the experimental data presented here and the computational data in the literature is that the setup of the QM/MM calculations froze the protein in the conformation observed for MhuD-haem-CN (PDB ID 4NL5), 10 but at least three other protein conformations exist. 13,14 The novel reactivity of the meso-hydroxyhaem form of MhuD could still be due to an electronic structure change, but this electronic structure change is not derived from substrate ruffling. Alternatively, the MhuD active site could catalyse meso-hydroxyhaem dioxygenation in a manner similar to cofactor-independent oxygenases, 35 with Streptomyces coelicolor ActVA-Orf6 being a particularly relevant precedent due to the structural homology of MhuD and ActVA-Orf6.…”

“…The Michaelis-Menten model is problematic for MhuD-catalysed haem oxygenation because: MhuD binds the haem substrate tightly with a Kd of 7.6 nM, 21 MhuD-catalysed haem oxygenation is a multi-step reaction, 18 and the enzyme is product-inhibited in vitro. 14 Thus, we have derived single-turnover kinetic expressions that are appropriate for the MhuD-catalysed reaction in this article. The enzyme catalysed conversion of haem to meso-hydroxyhaem is formally a monooxygenation, 18 and the influence of ruffling on the rate of this reaction has been assessed using an established assay.…”

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

“…[10][11][12] One explanation for the presence of two heme conformations in MhuD is that it is a consequence of the fact that at least four polypeptide conformational states exist. 10,13,14 Alternatively, from a heme electronic structure viewpoint, the ruffled conformation can be attributed to a hidden pseudo Jahn-Teller distortion arising from vibronic coupling of the 2 B2g and 2 A2u excited states of the planar species by a b1u vibration. 12,15,16 The populations of these conformations can be tuned by the F23W and W66F substitutions because these residues act like a vice on the heme substrate ( Figure 1).…”