Signal transduction and phosphoryl transfer by a FixL hybrid kinase with low oxygen affinity: importance of the vicinal PAS domain and receiver aspartate.

FixL is a prototype for heme-based sensors, multidomain proteins that typically couple a histidine protein kinase activity to a heme-binding domain for sensing of diatomic gases such as oxygen, carbon monoxide, and nitric oxide. Despite the relatively well-developed understanding of FixL, the importance of some of its domains has been unclear. To explore the impact of domain-domain interactions on oxygen sensing and signal transduction, we characterized and investigated Rhizobium etli hybrid sensor ReFixL.

Hell's Gate globin I: an acid and thermostable bacterial hemoglobin resembling mammalian neuroglobin.

Hell's Gate globin I (HGbI), a heme-containing protein structurally homologous to mammalian neuroglobins, has been identified from an acidophilic and thermophilic obligate methanotroph, Methylacidiphilum infernorum. HGbI has very high affinity for O(2) and shows barely detectable autoxidation in the pH range of 5.2-8.

Cyclic di-GMP activation of polynucleotide phosphorylase signal-dependent RNA processing.

The second messenger cyclic diguanylic acid (c-di-GMP) is implicated in key lifestyle decisions of bacteria, including biofilm formation and changes in motility and virulence. Some challenges in deciphering the physiological roles of c-di-GMP are the limited knowledge about the cellular targets of c-di-GMP, the signals that control its levels, and the proportion of free cellular c-di-GMP, if any. Here, we identify the target and the regulatory signal for a c-di-GMP-responsive Escherichia coli ribonucleoprotein complex.

An oxygen-sensing diguanylate cyclase and phosphodiesterase couple for c-di-GMP control.

A commonly observed coupling of sensory domains to GGDEF-class diguanylate cyclases and EAL-class phosphodiesterases has long suggested that c-di-GMP synthesizing and degrading enzymes sense environmental signals. Nevertheless, relatively few signal ligands have been identified for these sensors, and even fewer instances of in vitro switching by ligand have been demonstrated. Here we describe an Escherichia coli two-gene operon, dosCP, for control of c-di-GMP by oxygen.

Globins synthesize the second messenger bis-(3'-5')-cyclic diguanosine monophosphate in bacteria.

Globin-coupled sensors are heme-binding signal transducers in Bacteria and Archaea in which an N-terminal globin controls the activity of a variable C-terminal domain. Here, we report that BpeGReg, a globin-coupled diguanylate cyclase from the whooping cough pathogen Bordetella pertussis, synthesizes the second messenger bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP) upon oxygen binding. Expression of BpeGReg in Salmonella typhimurium enhances biofilm formation, while knockout of the BpeGReg gene of B.

Oxygen-sensing histidine-protein kinases: assays of ligand binding and turnover of response-regulator substrates.

Heme-based sensors are a recently discovered functional class of heme proteins that serve to detect physiological fluctuations in oxygen (O(2)), carbon monoxide (CO), or nitric oxide (NO). Many of these modular sensors detect heme ligands by coupling a histidine-protein kinase to a heme-binding domain. They typically bind O2, CO, and NO but respond only to one of these ligands.

DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis.

Exposure of Mycobacterium tuberculosis to hypoxia is known to alter the expression of many genes, including ones thought to be involved in latency, via the transcription factor DevR (also called DosR). Two sensory kinases, DosT and DevS (also called DosS), control the activity of DevR. We show that, like DevS, DosT contains a heme cofactor within an N-terminal GAF domain.

A memory of oxygen binding explains the dose response of the heme-based sensor FixL.

Bradyrhizobium japonicum FixL is a modular oxygen sensor that directs adaptations to hypoxia by coupling the status of a heme-binding domain to a histidine-protein kinase activity. The oxygen-bound form is the "off-state". The unliganded form is the "on-state" active kinase that phosphorylates a transcription factor, FixJ.

Determination and comparison of specific activity of the HIF-prolyl hydroxylases.

Hypoxia-inducible factor (HIF) is a transcriptional complex that is regulated by oxygen sensitive hydroxylation of its alpha subunits by the prolyl hydroxylases PHD1, 2 and 3. To better understand the role of these enzymes in directing cellular responses to hypoxia, we derived an assay to determine their specific activity in both native cell extracts and recombinant sources of enzyme. We show that all three are capable of high rates of catalysis, in the order PHD2=PHD3>PHD1, using substrate peptides derived from the C-terminal degradation domain of HIF-alpha subunits, and that each demonstrates similar and remarkable sensitivity to oxygen, commensurate with a common role in signaling hypoxia.

The HIF prolyl hydroxylase PHD3 is a potential substrate of the TRiC chaperonin.

Hypoxia-inducible factor-1 (HIF) is regulated by oxygen-dependent prolyl hydroxylation. Of the three HIF prolyl hydroxylases (PHD1, 2 and 3) identified, PHD3 exhibits restricted substrate specificity in vitro and is induced in different cell types by diverse stimuli. PHD3 may therefore provide an interface between oxygen sensing and other signalling pathways.

A distal arginine in oxygen-sensing heme-PAS domains is essential to ligand binding, signal transduction, and structure.

To evaluate the contributions of the G(beta)-2 arginine to signal transduction in oxygen-sensing heme-PAS domains, we replaced this residue with alanine in Bradyrhizobium japonicum FixL and examined the results on heme-domain structure, ligand binding, and kinase regulation. In the isolated R220A BjFixL heme-PAS domain, the iron-histidine bond was increased in length by 0.31 A, the heme flattened even without a ligand, and the interaction of a presumed regulatory loop (the FG loop) with the helix of heme attachment was weakened.

NPAS2: a gas-responsive transcription factor.

Neuronal PAS domain protein 2 (NPAS2) is a mammalian transcription factor that binds DNA as an obligate dimeric partner of BMAL1 and is implicated in the regulation of circadian rhythm. Here we show that both PAS domains of NPAS2 bind heme as a prosthetic group and that the heme status controls DNA binding in vitro. NPAS2-BMAL1 heterodimers, existing in either the apo (heme-free) or holo (heme-loaded) state, bound DNA avidly under favorably reducing ratios of the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate.

Ligand and oxidation-state specific regulation of the heme-based oxygen sensor FixL from Sinorhizobium meliloti.

Phosphorylation of the transcription factor RmFixJ is the key step in the hypoxic induction of Sinorhizobium meliloti nitrogen fixation genes. Oxygen regulates this process by binding reversibly to RmFixL, a heme protein kinase whose deoxy form catalyzes the phosphoryl transfer from ATP to RmFixJ. Here we present the first quantitative measure of the extent by which various heme ligands inhibit the turnover of RmFixJ to phospho-RmFixJ.