Iron Uptake Oxidoreductase (IruO) Uses a Flavin Adenine Dinucleotide Semiquinone Intermediate for Iron-Siderophore Reduction

Many pathogenic bacteria including <i>Staphylococcus aureus</i> use iron-chelating siderophores to acquire iron. Iron uptake oxidoreductase (IruO), a flavin adenine dinucleotide (FAD)-containing nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase from <i>S. aureus</i>, functions as a reductase for IsdG and IsdI, two paralogous heme degrading enzymes. Also, the gene encoding for IruO was shown to be required for growth of S. <i>aureus</i> on hydroxamate siderophores as a sole iron source. Here, we show that IruO binds the hydroxamate-type siderophores desferrioxamine B and ferrichrome A with low micromolar affinity and in the presence of NADPH, Fe­(II) was released. Steady-state kinetics of Fe­(II) release provides <i>k</i>_cat/<i>K</i>_m values in the range of 600 to 7000 M^–1 s^–1 for these siderophores supporting a role for IruO as a siderophore reductase in iron utilization. Crystal structures of IruO were solved in two distinct conformational states mediated by the formation of an intramolecular disulfide bond. A putative siderophore binding site was identified adjacent to the FAD cofactor. This site is partly occluded in the oxidized IruO structure consistent with this form being less active than reduced IruO. This reduction in activity could have a physiological role to limit iron release under oxidative stress conditions. Visible spectroscopy of anaerobically reduced IruO showed that the reaction proceeds by a single electron transfer mechanism through an FAD semiquinone intermediate. From the data, a model for single electron siderophore reduction by IruO using NADPH is described

Unique Heme-Iron Coordination by the Hemoglobin Receptor IsdB of <i>Staphylococcus aureus</i>

Iron is an essential requirement for life for nearly all organisms. The human pathogen <i>Staphylococcus aureus</i> is able to acquire iron from the heme cofactor of hemoglobin (Hb) released from lysed erythrocytes. IsdB, the predominant Hb receptor of <i>S. aureus</i>, is a cell wall-anchored protein that is composed of two NEAT domains. The N-terminal NEAT domain (IsdB-N1) binds Hb, and the C-terminal NEAT domain (IsdB-N2) relays heme to IsdA for transport into the cell. Here we present the 1.45 Å resolution X-ray crystal structure of the IsdB-N2–heme complex. While the structure largely conforms to the eight-strand β-sandwich fold seen in other NEAT domains such as IsdA-N and uses a conserved Tyr residue to coordinate heme-iron, a Met residue is also involved in iron coordination, resulting in a novel Tyr-Met hexacoordinate heme-iron state. The kinetics of the transfer of heme from IsdB-N2 to IsdA-N can be modeled as a two-step process. The rate of transfer of heme between the isolated NEAT domains (82 s^–1) was found to be similar to that measured for the full-length proteins. Replacing the iron coordinating Met with Leu did not abrogate high-affinity heme binding but did reduce the heme transfer rate constant by more than half. This unusual Met-Tyr heme coordination may also bestow properties on IsdB that help it to bind heme in different oxidation states or extract heme from hemoglobin

Hemoglobin Binding and Catalytic Heme Extraction by IsdB Near Iron Transporter Domains

The Isd (iron-regulated surface determinant) system is a multiprotein transporter that allows bacterium <i>Staphylococcus aureus</i> to take up iron from hemoglobin (Hb) during human infection. In this system, IsdB is a cell wall-anchored surface protein that contains two near iron transporter (NEAT) domains, one of which binds heme. IsdB rapidly extracts heme from Hb and transfers it to IsdA for relay into the bacterial cell. Using a series of recombinant IsdB constructs that included at least one NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity (<i>K</i>_D = 0.42 ± 0.05 μM) and to extract heme from Hb. Moreover, IsdB extracted heme only from oxidized metHb, although it also bound oxyHb and the Hb–CO complex. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed the transfer of heme from metHb to IsdA with a <i>K</i>_m for metHb of 0.75 ± 0.07 μN and a <i>k</i>_cat of 0.22 ± 0.01 s^–1. The latter is consistent with the transfer of heme from metHb to IsdB being the rate-limiting step. With both NEAT domains and the linker region present in a single contiguous polypeptide, high-affinity Hb binding was achieved, rapid heme uptake was observed, and multiple turnovers of heme extraction from metHb and transfer to IsdA were conducted, representing all known Hb–heme uptake functions of the full-length IsdB protein

Improved Manganese-Oxidizing Activity of DypB, a Peroxidase from a Lignolytic Bacterium

DypB, a dye-decolorizing peroxidase from the lignolytic soil bacterium <i>Rhodococcus jostii</i> RHA1, catalyzes the peroxide-dependent oxidation of divalent manganese (Mn²⁺), albeit less efficiently than fungal manganese peroxidases. Substitution of Asn246, a distal heme residue, with alanine increased the enzyme’s apparent <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m values for Mn²⁺ by 80- and 15-fold, respectively. A 2.2 Å resolution X-ray crystal structure of the N246A variant revealed the Mn²⁺ to be bound within a pocket of acidic residues at the heme edge, reminiscent of the binding site in fungal manganese peroxidase and very different from that of another bacterial Mn²⁺-oxidizing peroxidase. The first coordination sphere was entirely composed of solvent, consistent with the variant’s high <i>K</i>_m for Mn²⁺ (17 ± 2 mM). N246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and its solvent-extracted fractions. Two of the major degradation products were identified as 2,6-dimethoxybenzoquinone and 4-hydroxy-3,5-dimethoxybenzaldehyde, respectively. These results highlight the potential of bacterial enzymes as biocatalysts to transform lignin

Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis

<i>Staphylococcus aureus</i> assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from <i>O</i>-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5′-phosphate (PLP)-dependent enzyme with homology to <i>O</i>-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of <i>O</i>-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress

Repression of branched-chain amino acid synthesis in <i>Staphylococcus aureus</i> is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide

<div><p><i>Staphylococcus aureus</i> requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for <i>S</i>. <i>aureus</i> physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed <i>S</i>. <i>aureus</i> uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that <i>S</i>. <i>aureus</i> can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a <i>trans-</i>acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a <i>cis</i>-acting leucine-responsive attenuator, uncovering how <i>S</i>. <i>aureus</i> regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in <i>S</i>. <i>aureus</i>, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on <i>S</i>. <i>aureus</i> environmental adaptation and virulence via CodY.</p></div

Mutations identified in CodY.

<p>Mutations identified in CodY.</p

The absence of exogenous valine selects for mutations that inactivate CodY.

<p>A) Schematic representation of the mutations identified in CodY. B) Mutations identified are indicated on the CodY structure (PDB ID:5EY0) in yellow, except for the Ser180 to Pro mutation, which is indicated in green. CodY ligands, Ile and GTP, are coloured based on atomic composition. C) Strains were pre-grown in TSB to mid-exponential phase, then sub-cultured into TSB for 16 hr. Supernatants were collected and proteins were precipitated using TCA. Protein samples were normalized to the equivalent of 5 ODs and run on a 12% SDS-PAGE gel. D) Strains with unique mutations in <i>codY</i> (Val^Sup-2 carries an identical mutation to Val^Sup-4) were pre-grown in complete CDM to mid-exponential phase, then sub-cultured into either complete CDM or CDM with Val omitted. OD_600nm was read after 16 hr of growth. USA300 with a transposon insertion in <i>codY</i> (<i>codY</i>::ϕNΣ) was used for comparison. Val^Sup is abbreviated to Val^S. Data are the mean +/- SD of three biological replicates.</p

Ile is the predominant BCAA to regulate CodY activity on the <i>ilvD</i> promoter.

<p>WT <i>S</i>. <i>aureus</i> containing the <i>lux</i> reporter vector with either A) the partial <i>ilvD</i> promoter region (pGY<i>ilvD</i>^P::<i>lux</i>) or B) the complete <i>ilvD</i> promoter region (pGY<i>ilvD</i>^C::<i>lux</i>) was pre-grown in complete CDM to mid-exponential phase and then sub-cultured into either complete CDM or CDM with limiting concentrations of BCAAs, as indicated. Concentrations of Ile, Leu, and Val in complete CDM are 228 μM, 684 μM, 684 μM, respectively. Concentrations of Ile, Leu, and Val in limited media are 23 μM, 68 μM, and 68 μM, respectively. Luminescence values were read when cells reached mid-exponential phase and were normalized to the OD_600nm. Data are the mean of three biological replicates +/- SD. <i>S</i>. <i>aureus</i> strains with mutations in either <i>codY</i> (<i>codY</i>::ϕNΣ) or BCAA transporters and containing either C) the partial <i>ilvD</i> promoter region (pGY<i>ilvD</i>^P::<i>lux</i>) or D) the complete <i>ilvD</i> promoter region (pGY<i>ilvD</i>^C::<i>lux</i>) were pre-grown in complete CDM to mid-exponential phase and then sub-cultured into complete CDM. Luminescence values were read when cells reached mid-exponential phase and were normalized to the OD_600nm. Data are the mean of three biological replicates +/- SD. Data were analyzed by one-way ANOVA with Dunnet’s multiple comparisons test. *** <i>P</i> < 0.001.</p

Mutations identified in the 5’ UTR of <i>ilvD</i>.

<p>Mutations identified in the 5’ UTR of <i>ilvD</i>.</p