The affinity of MhuD for heme is consistent with a heme degrading function in vivo.
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ABSTRACT:
SUBMITTER: Thakuri B
PROVIDER: S-EPMC6529808 | biostudies-literature | 2018 Nov
REPOSITORIES: biostudies-literature
Publications
The affinity of MhuD for heme is consistent with a heme degrading function in vivo.
Thakuri Biswash B Graves Amanda B AB Chao Alex A Johansen Sommer L SL Goulding Celia W CW Liptak Matthew D MD
Metallomics : integrated biometal science 20181101 11
MhuD is a protein found in mycobacteria that can bind up to two heme molecules per protein monomer and catalyze the degradation of heme to mycobilin in vitro. Here the Kd1 for heme dissociation from heme-bound MhuD was determined to be 7.6 ± 0.8 nM and the Kd2 for heme dissocation from diheme-bound MhuD was determined to be 3.3 ± 1.1 μM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo. ...[more]
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Project description: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.
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