Giorgio Giardina

NO sensing in Pseudomonas aeruginosa: structure of the transcriptional regulator DNR.

All denitrifying bacteria can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels, controlling the expression of the denitrification gene clusters by redox signaling, mainly through transcriptional regulators belonging either to the DNR (dissimilative nitrate respiration regulator) or to the NnrR (nitrite and nitric oxide reductase regulator) subgroups of the FNR (fumarate and nitrate reductase regulatory protein)-CRP (cAMP receptor protein) superfamily. The NO dependence of the transcriptional activity of promoters regulated by these transcription factors has suggested that they may act as NO sensors in vivo. Despite great interest in the regulation of denitrification, which in Pseudomonas aeruginosa is strictly related to virulence, functional and structural characterization of these NO sensors is still lacking. Here we present the three-dimensional structure of the sensor domain of the DNR from P. aeruginosa at 2.1 A resolution. This is the first structure of a putative NO-sensing bacterial transcriptional regulator and reveals the presence of a large hydrophobic cavity that may be the cofactor binding site. Parallel spectroscopic evidence indicates that apo-DNR binds heme in vitro and that the heme-bound form reacts with carbon monoxide and NO, thus supporting the hypothesis that NO sensing involves gas binding to the ferrous heme. Preliminary experiments indicate that heterologous expression of the heme-containing DNR yields a protein able to bind DNA in vitro.

The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

Pseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix-turn-helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO.

Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to Group II decarboxylases

DOPA decarboxylase, the dimeric enzyme responsible for the synthesis of neurotransmitters dopamine and serotonin, is involved in severe neurological diseases such as Parkinson disease, schizophrenia, and depression. Binding of the pyridoxal-5′-phosphate (PLP) cofactor to the apoenzyme is thought to represent a central mechanism for the regulation of its activity. We solved the structure of the human apoenzyme and found it exists in an unexpected open conformation: compared to the pig kidney holoenzyme, the dimer subunits move 20 Å apart and the two active sites become solvent exposed. Moreover, by tuning the PLP concentration in the crystals, we obtained two more structures with different conformations of the active site. Analysis of three-dimensional data coupled to a kinetic study allows to identify the structural determinants of the open/close conformational change occurring upon PLP binding and thereby propose a model for the preferential degradation of the apoenzymes of Group II decarboxylases.

Nitrite and nitrite reductases: From molecular mechanisms to significance in human health and disease

Nitrite, previously considered physiologically irrelevant and a simple end product of endogenous nitric oxide (NO) metabolism, is now envisaged as a reservoir of NO to be activated in response to oxygen (O(2)) depletion. In the first part of this review, we summarize and compare the mechanisms of nitrite-dependent production of NO in selected bacteria and in eukaryotes. Bacterial nitrite reductases, which are copper or heme-containing enzymes, play an important role in the adaptation of pathogens to O(2) limitation and enable microrganisms to survive in the human body. In mammals, reduction of nitrite to NO under hypoxic conditions is carried out in tissues and blood by an array of metalloproteins, including heme-containing proteins and molybdenum enzymes. In humans, tissues play a more important role in nitrite reduction, not only because most tissues produce more NO than blood, but also because deoxyhemoglobin efficiently scavenges NO in blood. In the second part of the review, we outline the significance of nitrite in human health and disease and describe the recent advances and pitfalls of nitrite-based therapy, with special attention to its application in cardiovascular disorders, inflammation, and anti-bacterial defence. It can be concluded that nitrite (as well as nitrate-rich diet for long-term applications) may hold promise as therapeutic agent in vascular dysfunction and ischemic injury, as well as an effective compound able to promote angiogenesis.

Identification by Virtual Screening and In Vitro Testing of Human DOPA Decarboxylase Inhibitors

Dopa decarboxylase (DDC), a pyridoxal 5′-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinsons disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the “in vitro” activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with Ki values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with Ki values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a Ki value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

Investigating the allosteric regulation of YfiN from Pseudomonas aeruginosa: Clues from the structure of the catalytic domain

Pseudomonas aeruginosa is responsible for a plethora of biofilm mediated chronic infections among which cystic fibrosis pneumonia is the most frightening. The long-term survival strategy of P. aeruginosa in the patients lungs is based on a fine balance of virulence vs dormant states and on genetic adaptation, in order to select persistent phenotypes as the small colony variants (SCVs), which strongly correlate with antibiotic resistance and poor lung function. Recent studies have coupled SCV with increased levels of the signaling molecule cyclic di-GMP, and demonstrated the central role of the diguanylate cyclase YfiN, part of the tripartite signaling module YifBNR, in c-di-GMP dependent SCV regulation. YfiN, also called TpbB, is a multi-domain membrane enzyme connecting periplasmic stimuli to cytosolic c-di-GMP production by an allosteric inside-out signaling mechanism that, due to the lack of structural data, is still largely hypothetical. We have solved the crystal structure of the catalytic domain (GGDEF), and measured the enzymatic activity of the cytosolic portion in real-time by means of a newly developed method. Based on these results we demonstrate that, unlike other diguanylate cyclase, YfiN does not undergo product feedback inhibition, and that the presence of the HAMP domain is required for dimerization and catalysis. Coupling our structural and kinetic data with an in silico study we are now able to propose a model for the allosteric regulation of YfiN.

Probing the activity of diguanylate cyclases and c-di-GMP phosphodiesterases in real-time by CD spectroscopy

Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3′–5′ cyclic diguanylic acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3′–5′ cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.

C-di-GMP Hydrolysis by Pseudomonas aeruginosa HD-GYP Phosphodiesterases: Analysis of the Reaction Mechanism and Novel Roles for pGpG

In biofilms, the bacterial community optimizes the strategies to sense the environment and to communicate from cell to cell. A key player in the development of a bacterial biofilm is the second messenger c-di-GMP, whose intracellular levels are modulated by the opposite activity of diguanylate cyclases and phosphodiesterases. Given the huge impact of bacterial biofilms on human health, understanding the molecular details of c-di-GMP metabolism represents a critical step in the development of novel therapeutic approaches against biofilms. In this study, we present a detailed biochemical characterization of two c-di-GMP phosphodiesterases of the HD-GYP subtype from the human pathogen Pseudomonas aeruginosa, namely PA4781 and PA4108. Upstream of the catalytic HD-GYP domain, PA4781 contains a REC domain typical of two-component systems, while PA4108 contains an uncharacterized domain of unknown function. Our findings shed light on the activity and catalytic mechanism of these phosphodiesterases. We show that both enzymes hydrolyse c-di-GMP in a two-step reaction via the linear intermediate pGpG and that they produce GMP in vitro at a surprisingly low rate. In addition, our data indicate that the non-phosphorylated REC domain of PA4781 prevents accessibility of c-di-GMP to the active site. Both PA4108 and phosphorylated PA4781 are also capable to use pGpG as an alternative substrate and to hydrolyse it into GMP; the affinity of PA4781 for pGpG is one order of magnitude higher than that for c-di-GMP. These results suggest that these enzymes may not work (primarily) as genuine phosphodiesterases. Moreover, the unexpected affinity of PA4781 for pGpG may indicate that pGpG could also act as a signal molecule in its own right, thus further widening the c-di-GMP-related signalling scenario.

A dramatic conformational rearrangement is necessary for the activation of DNR from Pseudomonas aeruginosa. Crystal structure of wild‐type DNR

The opportunistic pathogen Pseudomonas aeruginosa can grow in low oxygen, because it is capable of anaerobic respiration using nitrate as a terminal electron acceptor (denitrification). An intermediate of the denitrification pathway is nitric oxide, a compound that may become cytotoxic at high concentration. The intracellular levels of nitric oxide are tightly controlled by regulating the expression of the enzymes responsible for its synthesis and degradation (nitrite and nitric oxide reductases). In this article, we present the crystallographic structure of the wild‐type dissimilative nitrate respiration regulator (DNR), a master regulator controlling expression of the denitrification machinery and a putative target for new therapeutic strategies. Comparison with other structures among the CRP‐FNR class of regulators reveals that DNR has crystallized in a conformation that has never been observed before. In particular, the sensing domain of DNR has undergone a rotation of more than 50° with respect to the other structures. This suggests that DNR may undergo an unexpected and very large conformational rearrangement on activation. Proteins 2009.

The Pseudomonas aeruginosa DNR transcription factor: light and shade of nitric oxide-sensing mechanisms

In response to environmental conditions, NO (nitric oxide) induces global changes in the cellular metabolism of Pseudomonas aeruginosa, which are strictly related to pathogenesis. In particular, at low oxygen tensions and in the presence of NO the denitrification alternative respiration is activated by a key regulator: DNR (dissimilative nitrate respiration regulator). DNR belongs to the CRP (cAMP receptor protein)-FNR (fumarate and nitrate reductase regulatory protein) superfamily of bacterial transcription factors. These regulators are involved in many different pathways and distinct activation mechanism seems to be operative in several cases. Recent results indicate that DNR is a haem protein capable of discriminating between NO and CO (carbon monoxide). On the basis of the available structural data, a suggested activation mechanism is discussed.