Catherine B. Poor

A New Oxidative Sensing and Regulation Pathway Mediated by the MgrA Homologue SarZ in Staphylococcus aureus

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses from the human pathogen Staphylococcus aureus. Among these, a thiol‐based oxidation sensing pathway through a global regulator MgrA controls the virulence and antibiotic resistance of the bacterium. Herein, we report a new thiol‐based oxidation sensing and regulation system that is mediated through a parallel global regulator SarZ. SarZ is a functional homologue of MgrA and is shown to affect the expression of ∼87 genes in S. aureus. It uses a key Cys residue, Cys‐13, to sense oxidative stress and to co‐ordinate the expression of genes involved in metabolic switching, antibiotic resistance, peroxide stress defence, virulence, and cell wall properties. The discovery of this SarZ‐mediated regulation, mostly independent from the MgrA‐based regulation, fills a missing gap of oxidation sensing and response in S. aureus.

Crystal structures of the reduced, sulfenic acid and mixed disulfide forms of sarz, a redox active global regulator in staphylococcus aureus

SarZ is a global transcriptional regulator that uses a single cysteine residue, Cys13, to sense peroxide stress and control metabolic switching and virulence in Staphylococcus aureus. SarZ belongs to the single-cysteine class of OhrR-MgrA proteins that play key roles in oxidative resistance and virulence regulation in various bacteria. We present the crystal structures of the reduced form, sulfenic acid form, and mixed disulfide form of SarZ. Both the sulfenic acid and mixed disulfide forms are structurally characterized for the first time for this class of proteins. The Cys13 sulfenic acid modification is stabilized through two hydrogen bonds with surrounding residues, and the overall DNA-binding conformation is retained. A further reaction of the Cys13 sulfenic acid with an external thiol leads to formation of a mixed disulfide bond, which results in an allosteric change in the DNA-binding domains, disrupting DNA binding. Thus, the crystal structures of SarZ in three different states provide molecular level pictures delineating the mechanism by which this class of redox active regulators undergoes activation. These structures help to understand redox-mediated virulence regulation in S. aureus and activation of the MarR family proteins in general.

Intramolecular amide bonds stabilize pili on the surface of bacilli

Gram-positive bacteria elaborate pili and do so without the participation of folding chaperones or disulfide bond catalysts. Sortases, enzymes that cut pilin precursors, form covalent bonds that link pilin subunits and assemble pili on the bacterial surface. We determined the x-ray structure of BcpA, the major pilin subunit of Bacillus cereus. The BcpA precursor encompasses 2 Ig folds (CNA2 and CNA3) and one jelly-roll domain (XNA) each of which synthesizes a single intramolecular amide bond. A fourth amide bond, derived from the Ig fold of CNA1, is formed only after pilin subunits have been incorporated into pili. We report that the domains of pilin precursors have evolved to synthesize a discrete sequence of intramolecular amide bonds, thereby conferring structural stability and protease resistance to pili.

Molecular mechanism and structure of the Saccharomyces cerevisiae iron regulator Aft2

Significance Iron is essential for eukaryotic cell survival but toxic at higher concentrations. In yeast, iron levels are tightly regulated by the transcriptional activators Aft1 and Aft2 (activators of ferrous transport), which activate iron-uptake genes when iron levels are low. We report the first crystal structure of DNA-bound Aft2 and show that Aft2 senses cellular iron levels via direct [2Fe-2S]-cluster binding, which promotes Aft2 dimerization and deactivation of the regulated genes. We further demonstrate that Aft2 acquires a [2Fe-2S] cluster from glutaredoxin-3 and Fe repressor of activation-2, two [2Fe-2S]-binding proteins with homologs in higher eukaryotes. This study unveils the molecular mechanism of the Aft family of iron-regulatory proteins and emphasizes the importance of Fe-S clusters in cellular iron sensing in eukaryotes. The paralogous iron-responsive transcription factors Aft1 and Aft2 (activators of ferrous transport) regulate iron homeostasis in Saccharomyces cerevisiae by activating expression of iron-uptake and -transport genes when intracellular iron is low. We present the previously unidentified crystal structure of Aft2 bound to DNA that reveals the mechanism of DNA recognition via specific interactions of the iron-responsive element with a Zn2+-containing WRKY-GCM1 domain in Aft2. We also show that two Aft2 monomers bind a [2Fe-2S] cluster (or Fe2+) through a Cys-Asp-Cys motif, leading to dimerization of Aft2 and decreased DNA-binding affinity. Furthermore, we demonstrate that the [2Fe-2S]-bridged heterodimer formed between glutaredoxin-3 and the BolA-like protein Fe repressor of activation-2 transfers a [2Fe-2S] cluster to Aft2 that facilitates Aft2 dimerization. Previous in vivo findings strongly support the [2Fe-2S] cluster-induced dimerization model; however, given the available evidence, Fe2+-induced Aft2 dimerization cannot be completely ruled out as an alternative Aft2 inhibition mechanism. Taken together, these data provide insight into the molecular mechanism for iron-dependent transcriptional regulation of Aft2 and highlight the key role of Fe-S clusters as cellular iron signals.

Directed Evolution of RebH for Site‐Selective Halogenation of Large Biologically Active Molecules

We recently characterized the substrate scope of wild-type RebH and proceeded to evolve variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. A substrate walking approach was used to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable the functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for the site-selective halogenation of biologically active compounds.

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.

Improving the stability and catalyst lifetime of the halogenase RebH by directed evolution.

We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3‐LR, with a Topt over 5 °C higher than that of wild‐type, and 3‐LSR, with a Tm 18 °C higher than that of wild‐type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non‐natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.

Gold(I)-Catalyzed Formation of 3-Pyrazolines through Cycloaddition of Diaziridine to Alkynes

This work reports the high-yield formation of pyrazoline derivatives mediated by gold(I) catalysts. The reaction utilizes a diaziridine, which has seen only limited usage in organic methodology. Mechanistic studies suggest a gold-mediated opening of the diazridine ring, alkyne insertion, and finally an intramolecular hydroamination to furnish the product.

Isopeptide bonds of the major pilin protein BcpA influence pilus structure and bundle formation on the surface of Bacillus cereus

Bacillus cereus strains elaborate pili on their surface using a mechanism of sortase‐mediated cross‐linking of major and minor pilus components. Here we used a combination of electron microscopy and atomic force microscopy to visualize these structures. Pili occur as single, double or higher order assemblies of filaments formed from monomers of the major pilin, BcpA, capped by the minor pilin, BcpB. Previous studies demonstrated that within assembled pili, four domains of BcpA – CNA1, CNA2, XNA and CNA3– each acquire intramolecular lysine‐asparagine isopeptide bonds formed via catalytic glutamic acid or aspartic acid residues. Here we showed that mutants unable to form the intramolecular isopeptide bonds in the CNA2 or CNA3 domains retain the ability to form pilus bundles. A mutant lacking the CNA1 isopeptide bond assembled deformed pilin subunits that failed to associate as bundles. X‐ray crystallography revealed that the BcpA variant Asp312Ala, lacking an aspartyl catalyst, did not generate the isopeptide bond within the jelly‐roll structure of XNA. The Asp312Ala mutant was also unable to form bundles and promoted the assembly of deformed pili. Thus, structural integrity of the CNA1 and XNA domains are determinants for the association of pili into higher order bundle structures and determine native pilus structure.

Structural and biochemical characterization of N5-carboxyaminoimidazole ribonucleotide synthetase and N5-carboxyaminoimidazole ribonucleotide mutase from Staphylococcus aureus.

With the rapid rise of methicillin-resistant Staphylococcus aureus infections, new strategies against S. aureus are urgently needed. De novo purine biosynthesis is a promising yet unexploited target, insofar as abundant evidence has shown that bacteria with compromised purine biosynthesis are attenuated. Fundamental differences exist within the process by which humans and bacteria convert 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). In bacteria, this transformation occurs through a two-step conversion catalyzed by PurK and PurE; in humans, it is mediated by a one-step conversion catalyzed by class II PurE. Thus, these bacterial enzymes are potential targets for selective antibiotic development. Here, the first comprehensive structural and biochemical characterization of PurK and PurE from S. aureus is presented. Structural analysis of S. aureus PurK reveals a nonconserved phenylalanine near the AIR-binding site that occupies the putative position of the imidazole ring of AIR. Mutation of this phenylalanine to isoleucine or tryptophan reduced the enzyme efficiency by around tenfold. The K(m) for bicarbonate was determined for the first time for a PurK enzyme and was found to be ∼18.8 mM. The structure of PurE is described in comparison to that of human class II PurE. It is confirmed biochemically that His38 is essential for function. These studies aim to provide foundations for future structure-based drug-discovery efforts against S. aureus purine biosynthesis.