Jason C. Grigg

Haem recognition by a Staphylococcus aureus NEAT domain

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem‐iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram‐positive bacteria. Many NEAT domain‐containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem‐binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo‐IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter‐like β‐sandwich fold with a large hydrophobic haem‐binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co‐ordinated solely by Tyr166. The phenol groups of Tyr166 and Tyr170 form an H‐bond that may function in regulating haem binding and release. An analysis of IsdA structure‐sequence alignments indicate that conservation of Tyr166 is a predictor of haem binding by NEAT domains.

Structural biology of heme binding in the Staphylococcus aureus Isd system

Iron is an absolute requirement for nearly all organisms, but most bacterial pathogens are faced with extreme iron-restriction within their host environments. To overcome iron limitation pathogens have evolved precise mechanisms to steal iron from host supplies. Staphylococcus aureus employs the iron-responsive surface determinant (Isd) system as its primary heme-iron uptake pathway. Hemoglobin or hemoglobin-haptoglobin complexes are bound by Near iron-Transport (NEAT) domains within cell surface anchored proteins IsdB or IsdH. Heme is stripped from the host proteins and transferred between NEAT domains through IsdA and IsdC to the membrane transporter IsdEF for internalization. Once internalized, heme can be degraded by IsdG or IsdI, thereby liberating iron for the organism. Most components of the Isd system have been structurally characterized to provide insight into the mechanisms of heme binding and transport. This review summarizes recent research on the Isd system with a focus on the structural biology of heme recognition.

Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus.

Iron is critical for virtually all forms of life. The production of high‐affinity iron chelators, siderophores, and the subsequent uptake of iron–siderophore complexes are a common strategy employed by microorganisms to acquire iron. Staphylococcus aureus produces siderophores but genetic information underlying their synthesis and transport is limited. Previous work implicated the sbn operon in siderophore synthesis and the sirABC operon in uptake. Here we characterize a second siderophore biosynthetic locus in S. aureus; the locus consists of four genes (in strain Newman these open reading frames are designated NWMN_2079–2082) which, together, are responsible for the synthesis and export of staphyloferrin A, a polycarboxylate siderophore. While deletion of the NWMN_2079–2082 locus did not affect iron‐restricted growth of S. aureus, strains bearing combined sbn and NWMN_2079–2082 locus deletions produced no detectable siderophore and demonstrated severely attenuated iron‐restricted growth. Adjacent to NWMN_2079–2082 resides the htsABC operon, encoding an ABC transporter previously implicated in haem acquisition. We provide evidence here that HtsABC, along with the FhuC ATPase, is required for the uptake of staphyloferrin A. The crystal structure of apo‐HtsA was determined and identified a large positively charged region in the substrate‐binding pocket, in agreement with a role in binding of anionic staphyloferrin A.

Heme Coordination by Staphylococcus aureus IsdE.

Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single α-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met78 and His229. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His229 is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.

Characterization of dye-decolorizing peroxidases from Rhodococcus jostii RHA1.

The soil bacterium Rhodococcus jostii RHA1 contains two dye-decolorizing peroxidases (DyPs) named according to the subfamily they represent: DypA, predicted to be periplasmic, and DypB, implicated in lignin degradation. Steady-state kinetic studies of these enzymes revealed that they have much lower peroxidase activities than C- and D-type DyPs. Nevertheless, DypA showed 6-fold greater apparent specificity for the anthraquinone dye Reactive Blue 4 (k(cat)/K(m) = 12800 ± 600 M(-1) s(-1)) than either ABTS or pyrogallol, consistent with previously characterized DyPs. By contrast, DypB showed the greatest apparent specificity for ABTS (k(cat)/K(m) = 2000 ± 100 M(-1) s(-1)) and also oxidized Mn(II) (k(cat)/K(m) = 25.1 ± 0.1 M(-1) s(-1)). Further differences were detected using electron paramagnetic resonance (EPR) spectroscopy: while both DyPs contained high-spin (S = (5)/(2)) Fe(III) in the resting state, DypA had a rhombic high-spin signal (g(y) = 6.32, g(x) = 5.45, and g(z) = 1.97) while DypB had a predominantly axial signal (g(y) = 6.09, g(x) = 5.45, and g(z) = 1.99). Moreover, DypA reacted with H(2)O(2) to generate an intermediate with features of compound II (Fe(IV)═O). By contrast, DypB reacted with H(2)O(2) with a second-order rate constant of (1.79 ± 0.06) × 10(5) M(-1) s(-1) to generate a relatively stable green-colored intermediate (t(1/2) ∼ 9 min). While the electron absorption spectrum of this intermediate was similar to that of compound I of plant-type peroxidases, its EPR spectrum was more consistent with a poorly coupled protein-based radical than with an [Fe(IV)═O Por(•)](+) species. The X-ray crystal structure of DypB, determined to 1.4 Å resolution, revealed a hexacoordinated heme iron with histidine and a solvent species occupying axial positions. A solvent channel potentially provides access to the distal face of the heme for H(2)O(2). A shallow pocket exposes heme propionates to the solvent and contains a cluster of acidic residues that potentially bind Mn(II). Insight into the structure and function of DypB facilitates its engineering for the improved degradation of lignocellulose.

Unique Heme-Iron Coordination by the Hemoglobin Receptor IsdB of Staphylococcus aureus.

Iron is an essential requirement for life for nearly all organisms. The human pathogen Staphylococcus aureus is able to acquire iron from the heme cofactor of hemoglobin (Hb) released from lysed erythrocytes. IsdB, the predominant Hb receptor of S. aureus, 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.

Distal Heme Pocket Residues of B-type Dye-decolorizing Peroxidase ARGININE BUT NOT ASPARTATE IS ESSENTIAL FOR PEROXIDASE ACTIVITY

Background: DypB, a Dyp-type peroxidase, oxidizes Mn(II) and transforms lignin. Results: DypB forms a stable Compound I that rapidly decays to Compound II in the D153A and N246A but is undetectable in the R244L variant. Conclusion: The requirement of Arg-244 but not Asp-153 to form Compound I indicates that DyPs modulate the peroxidative cycle differently than plant peroxidase. Significance: Understanding DyPs helps harness their biotechnological potential. DypB from Rhodococcus jostii RHA1 is a bacterial dye-decolorizing peroxidase (DyP) that oxidizes lignin and Mn(II). Three residues interact with the iron-bound solvent species in ferric DypB: Asn-246 and the conserved Asp-153 and Arg-244. Substitution of either Asp-153 or Asn-246 with alanine minimally affected the second order rate constant for Compound I formation (k1 ∼ 105 m−1s−1) and the specificity constant (kcat/Km) for H2O2. Even in the D153A/N246A double variant, these values were reduced less than 30-fold. However, these substitutions dramatically reduced the stability of Compound I (t½ ∼ 0.13 s) as compared with the wild-type enzyme (540 s). By contrast, substitution of Arg-244 with leucine abolished the peroxidase activity, and heme iron of the variant showed a pH-dependent transition from high spin (pH 5) to low spin (pH 8.5). Two variants were designed to mimic the plant peroxidase active site: D153H, which was more than an order of magnitude less reactive with H2O2, and N246H, which had no detectable peroxidase activity. X-ray crystallographic studies revealed that structural changes in the variants are confined to the distal heme environment. The data establish an essential role for Arg-244 in Compound I formation in DypB, possibly through charge stabilization and proton transfer. The principle roles of Asp-153 and Asn-246 appear to be in modulating the subsequent reactivity of Compound I. These results expand the range of residues known to catalyze Compound I formation in heme peroxidases.

The Staphylococcus aureus Siderophore Receptor HtsA Undergoes Localized Conformational Changes to Enclose Staphyloferrin A in an Arginine-rich Binding Pocket * □

Staphylococcus aureus uses several efficient iron acquisition strategies to overcome iron limitation. Recently, the genetic locus encoding biosynthetic enzymes for the iron chelating molecule, staphyloferrin A (SA), was determined. S. aureus synthesizes and secretes SA into its environment to scavenge iron. The membrane-anchored ATP binding cassette-binding protein, HtsA, receives the ferric-chelate for import into the cell. Recently, we determined the apoHtsA crystal structure, the first siderophore receptor from Gram-positive bacteria to be structurally characterized. Herein we present the x-ray crystal structure of the HtsA-ferric-SA complex. HtsA adopts a class III binding protein fold composed of separate N- and C-terminal domains bridged by a single α-helix. Recombinant HtsA can efficiently sequester ferric-SA from S. aureus culture supernatants where it is bound within the pocket formed between distinct N- and C-terminal domains. A basic patch composed mainly of six Arg residues contact the negatively charged siderophore, securing it within the pocket. The x-ray crystal structures from two different ligand-bound crystal forms were determined. The structures represent the first structural characterization of an endogenous α-hydroxycarboxylate-type siderophore-receptor complex. One structure is in an open form similar to apoHtsA, whereas the other is in a more closed conformation. The conformational change is highlighted by isolated movement of three loops within the C-terminal domain, a domain movement unique to known class III binding protein structures.

Improved manganese-oxidizing activity of DypB, a peroxidase from a lignolytic bacterium

DypB, a dye-decolorizing peroxidase from the lignolytic soil bacterium Rhodococcus jostii RHA1, catalyzes the peroxide-dependent oxidation of divalent manganese (Mn(2+)), albeit less efficiently than fungal manganese peroxidases. Substitution of Asn246, a distal heme residue, with alanine increased the enzymes apparent k(cat) and k(cat)/K(m) values for Mn(2+) by 80- and 15-fold, respectively. A 2.2 Å resolution X-ray crystal structure of the N246A variant revealed the Mn(2+) 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 Mn(2+)-oxidizing peroxidase. The first coordination sphere was entirely composed of solvent, consistent with the variants high K(m) for Mn(2+) (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.

Iron-Coordinating Tyrosine Is a Key Determinant of NEAT Domain Heme Transfer.

In humans, heme iron is the most abundant iron source, and bacterial pathogens such as Staphylococcus aureus acquire it for growth. IsdB of S. aureus acquires Fe(III)-protoporphyrin IX (heme) from hemoglobin for transfer to IsdC via IsdA. These three cell-wall-anchored Isd (iron-regulated surface determinant) proteins contain conserved NEAT (near iron transport) domains. The purpose of this work was to delineate the mechanism of heme binding and transfer between the NEAT domains of IsdA, IsdB, and IsdC using a combination of structural and spectroscopic studies. X-ray crystal structures of IsdA NEAT domain (IsdA-N1) variants reveal that removing the native heme-iron ligand Tyr166 is compensated for by iron coordination by His83 on the distal side and that no single mutation of distal loop residues is sufficient to perturb the IsdA-heme complex. Also, alternate heme-iron coordination was observed in structures of IsdA-N1 bound to reduced Fe(II)-protoporphyrin IX and Co(III)-protoporphyrin IX. The IsdA-N1 structural data were correlated with heme transfer kinetics from the NEAT domains of IsdB and IsdC. We demonstrated that the NEAT domains transfer heme at rates comparable to full-length proteins. The second-order rate constant for heme transfer from IsdA-N1 was modestly affected (<2-fold) by the IsdA variants, excluding those at Tyr166. Substituting Tyr166 with Ala or Phe changed the reaction mechanism to one with two observable steps and decreased observed rates >15-fold (to 100-fold excess IsdC). We propose a heme transfer model wherein NEAT domain complexes pass heme iron directly from an iron-coordinating Tyr of the donor protein to the homologous Tyr residues of the acceptor protein.