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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 IX? (?BV) 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 ?BV as a proxy to study the IsdG-type protein-product complex. First, we show that ?BV has a nanomolar affinity for MhuD and the R26S variant. Second, we determined the MhuD-R26S-?BV complex structure to 2.5 Å, which reveals two notable features: (1) two ?BV molecules bound per active site and (2) a novel ?-helix (?3) that was not observed in previous MhuD-heme structures. Finally, through molecular dynamics simulations, we show that ?3 is stable with the proximal ?BV 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.

SUBMITTER: Chao A

PROVIDER: S-EPMC7045704 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Structure of a <i>Mycobacterium tuberculosis</i> Heme-Degrading Protein, MhuD, Variant in Complex with Its Product.

Chao Alex A Burley Kalistyn H KH Sieminski Paul J PJ de Miranda Rodger R Chen Xiaorui X Mobley David L DL Goulding Celia W CW

Biochemistry 20191106 46

<i>Mycobacterium tuberculosis</i> (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-ty ...[more]

PMID: 31638374

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Project description:Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the ?-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the ?- and ?-meso carbons, whereas the spin density distribution may favor selective oxygenation of the ?-meso carbon.

Dataset Information

Structure of a Mycobacterium tuberculosis Heme-Degrading Protein, MhuD, Variant in Complex with Its Product.

Publications

Structure of a <i>Mycobacterium tuberculosis</i> Heme-Degrading Protein, MhuD, Variant in Complex with Its Product.

Similar Datasets

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