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.

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Pseudomonas aeruginosa is an opportunistic, Gram‐negative pathogen that is resistant to many classes of antibiotics. One pathway leading to resistance to cationic antimicrobial peptides like colistin is upregulation of the arnBCADTEF operon. With the biosynthetic enzymes encoded in this pathway, P. aeruginosa modifies its outer membrane to decrease its net negative charge. This neutralization of charge lessens the driving force for electrostatic interactions with positively charged antibiotics such as colistin, hindering their entry into the cell. The first committed step of this pathway is catalyzed by the decarboxylase domain of ArnA in which UDP‐glucuronic acid undergoes a redox reaction with NAD+. Due to its role in the arnB pathway of antibiotic resistance, we seek to characterize the kinetics of ArnA using in vitro biochemical studies. To do this we measured the change in absorbance at 340 nm allowing us to calculate the reduction of NAD+ to NADH. These data were analyzed to produce the Michaelis‐Menten kinetics of ArnA from P. aeruginosa. Elucidating the kinetics of ArnA will enable analysis of small molecule inhibitors for changes in the activity of this enzyme. This knowledge will enable future work to design strategies to inhibit ArnA and thus block the resistance pathway.Support or Funding InformationNational Institutes of Health (NIH) Grant # 5SC3GM118199This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In this study we investigated the effects of the branched chain alpha-ketoacids accumulating in maple syrup urine disease (MSUD) on the concentrations of the high molecular weight neurofilament subunit (NF-H) associated with the cytoskeletal fraction of the cerebral cortex of 12-day-old rats. Cortical slices were incubated with alpha-ketoisocaproic acid (KIC), alpha-keto beta-methylvaleric acid (KMV) and alpha-ketoisovaleric acid (KIV) at concentrations ranging from 0.5 to 1.0 mM. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated and total NF-H was analyzed by immunoblotting. The in vitro 32P incorporation into NF-H was also determined. Results showed that treatment of tissue slices induced with KMV increased Triton-insoluble phosphorylated NF-H immunoreactivity, with no alteration in total NF-H immunoreactivity. Furthermore, KIC treatment drastically increased the total amount of NF-H, whereas KIV did not change either phosphorylated or total NF-H immunoreactivity. KMV also increased the in vitro 32P incorporation into NF-H, confirming the highly phosphorylated NF-H levels detected in the immunoblot. These findings demonstrate that KIC and KMV alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction. Therefore we may suggest that cytoskeletal disorganization may be one of the factors associated with the neurodegeneration characteristic of MSUD disease.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), results in 10 million infections and 1.5 million deaths annually. Current TB treatments are typically a cocktail of up to five antibiotics that needs to be administered for a 9‐month duration; unfortunately, several of these drugs illicit severe side effects. These long, harsh treatments lead to patient non‐compliance resulting in a rise in multiple‐drug resistant Mtb strains ‐ MDR‐TB. Due to these factors new treatment strategies are needed. As host iron acquisition is essential for Mtb’s survival, elucidating key players in the Mtb iron uptake pathways may yield good drug targets. Mtb predominately acquires host iron by the siderophore‐mediated uptake pathway, in which small molecules with high affinity for iron are secreted to scavenge for host iron. We seek to shed light on the mechanism by which ferric‐siderophores are transported into the Mtb cytosol. At present there is little known about the proteins required to shuttle ferric‐siderophores across the outer membrane, cell‐wall environment, and periplasmic space to the inner membrane. There are two putative Mtb periplasmic binding proteins (PBPs), FecB and FecB2, and we hypothesize that one or both of these PBPs shuttle ferric‐siderophores across the periplasmic space. To test this, we examined the affinity of FecB and FecB2 for the Mtb secreted ferric‐siderophore, ferric‐carboxymycobactin (Fe‐cMB), by tryptophan fluorescence quenching titration assays. The affinity of FecB for Fe‐cMB was in the high nanomolar range (Kd = 355 ± 146 nM); in contrast, the affinity of FecB2 was in the high micromolar range. This result suggests that FecB is involved in transporting Fe‐cMB across the periplasmic space. To further probe the FecB residues involved in Fe‐cMB binding, we carried out a comprehensive mutational analysis. In an attempt to decipher the protein‐protein interaction network of FecB, we utilized co‐immunopreciptation (co‐IP) of FLAG‐tagged FecB in the non‐pathogenic Mtb model organism Mycobacterium smegmatis, followed by mass spectrometry analysis. The co‐IP FLAG‐FecB experiments resulted in identification of known proteins in the import of Fe‐cMB and surprisingly, also proteins thought to be involved in the export of apo‐siderophores. Together, these data clearly place FecB in the siderophore‐mediated iron‐uptake pathway, and will be discussed in more detail. Finally, this work has broadened our understanding of the Mtb iron acquisition pathway, which may lead to the identification of new therapeutic targets.