H-NOX Influences Biofilm Formation, Central Metabolism, and Quorum Sensing in .

The transition from planktonic to biofilm growth in bacteria is often accompanied by greater resistance to antibiotics and other stressors, as well as distinct alterations in physical traits, genetic activity, and metabolic restructuring. In many species, the heme nitric oxide/oxygen binding proteins (H-NOX) play an important role in this process, although the signaling mechanisms and pathways in which they participate are quite diverse and largely unknown. In , deletion of the gene results in a severe biofilm-deficient phenotype.

Influence of the Heme Nitric Oxide/Oxygen Binding Protein (H-NOX) on Cell Cycle Regulation in Caulobacter crescentus.

The ability of an organism to respond to environmental changes is paramount to survival across a range of conditions. The bacterial heme nitric oxide/oxygen binding proteins (H-NOX) are a family of biofilm-regulating gas sensors that enable bacteria to respond accordingly to the cytotoxic molecule nitric oxide. By interacting with downstream signaling partners, H-NOX regulates the production of the bacterial secondary messenger cyclic diguanylate monophosphate (c-di-GMP) to influence biofilm formation.

Structure and Function of the Zinc Binding Protein ZrgA from .

ATP binding cassette (ABC) transporters are the primary means by which bacteria acquire trace elements from the environment. They rely on solute binding proteins (SBPs) to bind the relevant substrate and deliver it to the integral membrane permease for ATP-powered import into the cytoplasm. SBPs of cluster A-I are known to facilitate the transport of essential metals zinc, manganese, and iron, and many have been characterized to date.

Contributions of Conformational Flexibility to High-Affinity Zinc Binding in the Solute Binding Protein AztC.

Bacteria rely on ATP binding cassette (ABC) transporters for the import of various nutrients. Bacterial ABC importers utilize an extracellular solute binding protein (SBP) to bind the substrate with high affinity and specificity and deliver it to the membrane permease for transport. The essential metals iron, manganese, and zinc are bound and transported by the cluster A-I SBPs.

Specificity of Interactions between Components of Two Zinc ABC Transporters in .

Bacterial ATP binding cassette (ABC) transporters mediate the influx of numerous substrates. The cluster A-I ABC transporters are responsible for the specific uptake of the essential metals zinc, manganese or iron, making them necessary for survival in metal-limited environments, which for pathogens include the animal host. In , there are two zinc ABC transporter systems: ZnuABC and AztABCD with apparently redundant functions under zinc-limited conditions.

Structural Features Mediating Zinc Binding and Transfer in the AztABCD Zinc Transporter System.

Many bacteria require ATP binding cassette (ABC) transporters for the import of the essential metal zinc from limited environments. These systems rely on a periplasmic or cell-surface solute binding protein (SBP) to bind zinc with high affinity and specificity. AztABCD is one such zinc transport system recently identified in a large group of diverse bacterial species.

Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase.

The quinoprotein glycine oxidase from the marine bacterium (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis.

Structural insights into the mechanism of oxidative activation of heme-free H-NOX from Vibrio cholerae.

Bacterial heme nitric oxide/oxygen (H-NOX) domains are nitric oxide (NO) or oxygen sensors. This activity is mediated through binding of the ligand to a heme cofactor. However, H-NOX from Vibrio cholerae (Vc H-NOX) can be easily purified in a heme-free state that is capable of reversibly responding to oxidation, suggesting a heme-independent function as a redox sensor.

Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase.

PlGoxA from is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation.

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins.

Zinc acquisition from limited environments is critical for bacterial survival and pathogenesis. AztD has been identified as a periplasmic or cell surface zinc-binding protein in numerous bacterial species. In , AztD can transfer zinc directly to AztC, the solute binding protein for a zinc-specific ATP-binding cassette transporter system, suggesting a role in zinc acquisition and homeostasis.

Structural and Spectroscopic Characterization of a Product Schiff Base Intermediate in the Reaction of the Quinoprotein Glycine Oxidase, GoxA.

The LodA-like proteins make up a recently identified family of enzymes that rely on a cysteine tryptophylquinone cofactor for catalysis. They differ from other tryptophylquinone enzymes in that they are oxidases rather than dehydrogenases. GoxA is a member of this family that catalyzes the oxidative deamination of glycine.

Two ABC Transporters and a Periplasmic Metallochaperone Participate in Zinc Acquisition in Paracoccus denitrificans.

Bacteria must acquire the essential element zinc from extremely limited environments, and this function is performed largely by ATP binding cassette (ABC) transporters. These systems rely on a periplasmic or extracellular solute binding protein (SBP) to bind zinc specifically with a high affinity and deliver it to the membrane permease for import into the cytoplasm. However, zinc acquisition systems in bacteria may be more complex, involving multiple transporters and other periplasmic or extracellular zinc binding proteins.

Diversity of structures, catalytic mechanisms and processes of cofactor biosynthesis of tryptophylquinone-bearing enzymes.

Tryptophyquinone-bearing enzymes contain protein-derived cofactors formed by posttranslational modifications of Trp residues. Tryptophan tryptophylquinone (TTQ) is comprised of a di-oxygenated Trp residue, which is cross-linked to another Trp residue. Cysteine tryptophylquinone (CTQ) is comprised of a di-oxygenated Trp residue, which is cross-linked to a Cys residue.

Structure and Enzymatic Properties of an Unusual Cysteine Tryptophylquinone-Dependent Glycine Oxidase from Pseudoalteromonas luteoviolacea.

Glycine oxidase from Pseudoalteromonas luteoviolacea (PlGoxA) is a cysteine tryptophylquinone (CTQ)-dependent enzyme. Sequence analysis and phylogenetic analysis place it in a newly designated subgroup (group IID) of a recently identified family of LodA-like proteins, which are predicted to possess CTQ. The crystal structure of PlGoxA reveals that it is a homotetramer.

Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets.

Zinc-Dependent Transcriptional Regulation in .

Zinc homeostasis is critical for bacterial survival and is mediated largely at the transcriptional level by the regulation of zinc uptake and efflux genes. Here we use RNA-seq to assess transcriptional changes as a result of zinc limitation in the denitrifying bacterium . The results identify the differential expression of 147 genes, most of which were upregulated in zinc-depleted medium.

Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae.

Heme nitric oxide/oxygen (H-NOX)-binding proteins act as nitric oxide (NO) sensors among various bacterial species. In several cases, they act to mediate communal behavior such as biofilm formation, quorum sensing, and motility by influencing the activity of downstream signaling proteins such as histidine kinases (HisKa) in a NO-dependent manner. An H-NOX/HisKa regulatory circuit was recently identified in Vibrio cholerae, and the H-NOX protein has been spectroscopically characterized.

Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS.

Mycobacterium tuberculosis DosS is critical for the induction of M. tuberculosis dormancy genes in response to nitric oxide (NO), carbon monoxide (CO), or hypoxia. These environmental stimuli, which are sensed by the DosS heme group, result in autophosphorylation of a DosS His residue, followed by phosphotransfer to an Asp residue of the response regulator DosR.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography.

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected.

AztD, a Periplasmic Zinc Metallochaperone to an ATP-binding Cassette (ABC) Transporter System in Paracoccus denitrificans.

Bacterial ATP-binding cassette (ABC) transporters of transition metals are essential for acquisition of necessary elements from the environment. A large number of Gram-negative bacteria, including human pathogens, have a fourth conserved gene of unknown function adjacent to the canonical permease, ATPase, and solute-binding protein (SBP) genes of the AztABC zinc transporter system. To assess the function of this putative accessory factor (AztD) from Paracoccus denitrificans, we have analyzed its transcriptional regulation, metal binding properties, and interaction with the SBP (AztC).

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans.

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn(2+) and Mn(2+) from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn(2+) or Mn(2+) specificity remain ambiguous.

A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36.

The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36.

Site-directed mutagenesis of Gln103 reveals the influence of this residue on the redox properties and stability of MauG.

The diheme enzyme MauG catalyzes a six-electron oxidation that is required for the posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived cofactor, tryptophan tryptophylquinone (TTQ). Crystallographic and computational studies have implicated Gln103 in stabilizing the Fe(IV)═O moiety of the bis-Fe(IV) state by hydrogen bonding. The role of Gln103 was probed by site-directed mutagenesis.

Oxidative damage in MauG: implications for the control of high-valent iron species and radical propagation pathways.

The di-heme enzyme MauG catalyzes the oxidative biosynthesis of a tryptophan tryptophylquinone cofactor on a precursor of the enzyme methylamine dehydrogenase (preMADH). Reaction of H2O2 with the diferric form of MauG, or reaction of O2 with diferrous MauG, forms the catalytic intermediate known as bis-Fe(IV), which acts as the key oxidant during turnover. The site of substrate oxidation is more than 40 Å from the high-spin heme iron where H2O2 initially reacts, and catalysis relies on radical hopping through an interfacial residue, Trp199 of MauG.

Carboxyl group of Glu113 is required for stabilization of the diferrous and bis-Fe(IV) states of MauG.

The diheme enzyme MauG catalyzes a six-electron oxidation required for post-translational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies have implicated Glu113 in the formation of the bis-Fe(IV) state of MauG, in which one heme is Fe(IV)═O and the other is Fe(IV) with His-Tyr axial ligation. An E113Q mutation had no effect on the structure of MauG but significantly altered its redox properties.