Cyclic AMP-Mediated Inhibition of Cholesterol Catabolism in Mycobacterium tuberculosis by the Novel Drug Candidate GSK2556286.

Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis, shortens treatment in rodents relative to standard of care drugs. Moreover, GSK286 can replace linezolid in the three-drug, Nix-TB regimen. Given its therapeutic potential, we sought to better understand the mechanism of action of GSK286. The compound blocked growth of M. tuberculosis in cholesterol media and increased intracellular cAMP levels ~50-fold. GSK286 did not inhibit growth of an rv1625c transposon mutant in cholesterol media and did not induce cyclic AMP (cAMP) production in this mutant, suggesting that the compound acts on this adenylyl cyclase. GSK286 also induced cAMP production in Rhodococcus jostii RHA1, a cholesterol-catabolizing actinobacterium, when Rv1625c was heterologously expressed. However, these elevated levels of cAMP did not inhibit growth of R. jostii RHA1 in cholesterol medium. Mutations in rv1625c conferred cross-resistance to GSK286 and the known Rv1625c agonist, mCLB073. Metabolic profiling of M. tuberculosis cells revealed that elevated cAMP levels, induced using either an agonist or a genetic tool, did not significantly affect pools of steroid metabolites in cholesterol-incubated cells. Finally, the inhibitory effect of agonists was not dependent on the N-acetyltransferase MtPat. Together, these data establish that GSK286 is an Rv1625c agonist and sheds light on how cAMP signaling can be manipulated as a novel antibiotic strategy to shorten TB treatments. Nevertheless, the detailed mechanism of action of these compounds remains to be elucidated.

Bacterial catabolism of acetovanillone, a lignin-derived compound.

Bacterial catabolic pathways have considerable potential as industrial biocatalysts for the valorization of lignin, a major component of plant-derived biomass. Here, we describe a pathway responsible for the catabolism of acetovanillone, a major component of several industrial lignin streams. Rhodococcus rhodochrous GD02 was previously isolated for growth on acetovanillone. A high-quality genome sequence of GD02 was generated. Transcriptomic analyses revealed a cluster of eight genes up-regulated during growth on acetovanillone and 4-hydroxyacetophenone, as well as a two-gene cluster up-regulated during growth on acetophenone. Bioinformatic analyses predicted that the hydroxyphenylethanone (Hpe) pathway proceeds via phosphorylation and carboxylation, before ß-elimination yields vanillate from acetovanillone or 4-hydroxybenzoate from 4-hydroxyacetophenone. Consistent with this prediction, the kinase, HpeHI, phosphorylated acetovanillone and 4-hydroxyacetophenone. Furthermore, HpeCBA, a biotin-dependent enzyme, catalyzed the ATP-dependent carboxylation of 4-phospho-acetovanillone but not acetovanillone. The carboxylase's specificity for 4-phospho-acetophenone (kcat/KM = 34 ± 2 mM-1 s-1) was approximately an order of magnitude higher than for 4-phospho-acetovanillone. HpeD catalyzed the efficient dephosphorylation of the carboxylated products. GD02 grew on a preparation of pine lignin produced by oxidative catalytic fractionation, depleting all of the acetovanillone, vanillin, and vanillate. Genomic and metagenomic searches indicated that the Hpe pathway occurs in a relatively small number of bacteria. This study facilitates the design of bacterial strains for biocatalytic applications by identifying a pathway for the degradation of acetovanillone.

Staphylococcus aureus heme and siderophore-iron acquisition pathways.

Staphylococcus aureus is a versatile opportunistic human pathogen. Infection by this bacterium requires uptake of iron from the human host, but iron is highly restricted in this environment. Staphylococcus aureus iron sufficiency is achieved primarily through uptake of heme and high-affinity iron chelators, known as siderophores. Two siderophores (staphyloferrins) are produced and secreted by S. aureus into the extracellular environment to capture iron. Staphylococcus aureus expresses specific uptake systems for staphyloferrins and more general uptake systems for siderophores produced by other microorganisms. The S. aureus heme uptake system uses highly-specific cell surface receptors to extract heme from hemoglobin and hemoglobin-haptoglobin complexes for transport into the cytoplasm where it is degraded to liberate iron. Initially thought to be independent systems, recent findings indicate that these iron uptake pathways intersect. IruO is a reductase that releases iron from heme and some ferric-siderophores. Moreover, multifunctional SbnI produces a precursor for staphyloferrin B biosynthesis, and also binds heme to regulate expression of the staphyloferrin B biosynthesis pathway. Intersection of the S. aureus iron uptake pathways is hypothesized to be important for rapid adaptation to available iron sources. Components of the heme and siderophore uptake systems are currently being targeted in the development of therapeutics against S. aureus.

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

Staphylococcus aureus 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 O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-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.

Structures of Large RNAs and RNA-Protein Complexes: Toward Structure Determination of Riboswitches.

Riboswitches are widespread and important regulatory elements. They are typically present in the mRNA of the gene under their regulation, where they form complex three-dimensional structures that can bind an effector and regulate either transcription or translation of the mRNA. Structural biology has been essential to our understanding of their ligand recognition and conformational switching mechanisms, but riboswitch determination presents several important complications. Overcoming these challenges requires a synergistic approach using rational design of the constructs and supporting methods to biochemically validate the designs and resulting structures.

The B-type channel is a major route for iron entry into the ferroxidase center and central cavity of bacterioferritin.

Bacterioferritin is a bacterial iron storage and detoxification protein that is capable of forming a ferric oxyhydroxide mineral core within its central cavity. To do this, iron must traverse the bacterioferritin protein shell, which is expected to occur through one or more of the channels through the shell identified by structural studies. The size and negative electrostatic potential of the 24 B-type channels suggest that they could provide a route for iron into bacterioferritin. Residues at the B-type channel (Asn-34, Glu-66, Asp-132, and Asp-139) of E. coli bacterioferritin were substituted to determine if they are important for iron core formation. A significant decrease in the rates of initial oxidation of Fe(II) at the ferroxidase center and subsequent iron mineralization was observed for the D132F variant. The crystal structure of this variant shows that substitution of residue 132 with phenylalanine caused a steric blockage of the B-type channel and no other material structural perturbation. We conclude that the B-type channel is a major route for iron entry into both the ferroxidase center and the iron storage cavity of bacterioferritin.

Common themes and differences in SAM recognition among SAM riboswitches.

The recent discovery of short cis-acting RNA elements termed riboswitches has caused a paradigm shift in our understanding of genetic regulatory mechanisms. The three distinct superfamilies of S-adenosyl-l-methionine (SAM) riboswitches are the most commonly found riboswitch classes in nature. These RNAs represent three independent evolutionary solutions to achieve specific SAM recognition. This review summarizes research on 1) modes of gene regulatory mechanisms, 2) common themes and differences in ligand recognition, and 3) ligand-induced conformational dynamics among SAM riboswitch families. The body of work on the SAM riboswitch families constitutes a useful primer to the topic of gene regulatory RNAs as a whole. This article is part of a Special Issue entitled: Riboswitches.

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.

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 ß-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.