The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.The pyridine nucleotide cycle (PNC) 3 is a network of biochemical transformations that allow cells to recycle the by-products of endogenous NAD consumption back to the coenzyme and to salvage the available pyridine bases, nucleosides, and nucleotides as NAD precursors. The importance of NAD regeneration through recycling pathways is emphasized by the occurrence of an intense nonredox NAD consumption as suggested by the rapid turnover of the coenzyme pool within the cell (1). In bacteria, the pyridine by-products of the NADconsuming enzymes NMN and Nm can be recycled back to NAD through the PNC depicted in Fig. 1 (2, 3). Briefly, Nm can be converted to NAD through two different routes. The most commonly occurring pathway is initiated by Nm deamidation to Na, followed by Na conversion to NaMN, NaMN adenylation to NaAD, and NaAD amidation to NAD. The last three reactions comprise the so-called Preiss-Handler pathway (4, 5). The second Nm recycling route is a relatively rare, nondeamidated pathway, whereby Nm is directly phosphoribosylated to NMN and NMN is then adenylated to NAD. NMN can be recycled back to NAD through two pathways shown to be functional in Escherichia coli and Salmonella typhymurium (6): the predominant route, PNC IV, proceeds via NMN deamidation to NaMN, which is then converted to NAD by entering the PreissHandler pathway; the alternati...

HdeA protein is a small, ATP-independent, acid stress chaperone that undergoes a dimerto-monomer transition in acidic environments. The HdeA monomer binds a broad range of proteins to prevent their acid-induced aggregation. To understand better HdeA's function and mechanism, we perform constant-pH molecular dynamics simulations (CPHMD) to elucidate the details of the HdeA dimer dissociation process. First the pKa values of all the acidic titratable groups in HdeA are obtained and reveal a large pKa shift only for Glu(37). However, the pH-dependent monomer charge exhibits a large shift from −4 at pH > 6 to +6 at pH = 2.5, suggesting the dramatic change in charge on each monomer may drive dissociation. By combining the CPHMD approach with umbrella sampling, we demonstrate a significant stability decrease of the HdeA dimer when the environmental pH changes from 4.0 to 3.5, and identify the key acidic residue – lysine interactions responsible for the observed pH sensing in HdeA chaperon activity function.

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Edited by Miguel De la RosaKeywords: NMN deamidase Pyridine nucleotide Catalytic dyad Amidohydrolase Site-directed mutagenesis a b s t r a c t NMN deamidase (PncC) is a bacterial enzyme involved in NAD biosynthesis. We have previously demonstrated that PncC is structurally distinct from other known amidohydrolases. Here, we extended PncC characterization by mutating all potential catalytic residues and assessing their individual roles in catalysis through kinetic analyses. Inspection of these residues' spatial arrangement in the active site, allowed us to conclude that PncC is a serine-amidohydrolase, employing a Ser/Lys dyad for catalysis. Analysis of the PncC structure in complex with a modeled NMN substrate supported our conclusion, and enabled us to propose the catalytic mechanism.

The PcF Toxin Family (Pfam 09461) includes the characterized phytotoxic protein PcF from Phytophthora cactorum, as well as several predicted protein effectors from other Phytophthora species recently identified by comparative genomics. Here we provide first evidence that such 'putatives', recombinantly expressed in bacteria and purified to homogeneity, similarly to PcF, can trigger defense-related responses on tomato, that is leaf withering and phenylalanine ammonia lyase induction, although with various degrees of effectiveness. In addition, structural prediction by computer-aided homology modeling and subsequent structural/functional comparison after rational engineering of the disulfide-structured protein fold by site-directed mutagenesis, highlighted the surface-exposed conserved amino acid stretch SK(E/C)C as a possible structural determinant responsible for the differential phytotoxicity within this family of cognate protein effectors.

Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the formation of NAD by means of nucleophilic attack by 5 0 -phosphoryl of NMN on the a-phosphoryl group of ATP. Humans possess three NMNAT isozymes (NMNAT1, NMNAT2, and NMNAT3) that differ in size and sequence, gene expression pattern, subcellular localization, oligomeric state and catalytic properties. Of these, NMNAT2, the least abundant isozyme, is the only one whose much-needed crystal structure has not been solved as yet. To fill this gap, we used the crystal structures of human NMNAT1 and NMNAT3 as templates for homology-based structural modeling of NMNAT2, and the resulting raw structure was then refined by molecular dynamics simulations in a water box to obtain a model of the final folded structure. We investigated the importance of NMNAT2's central domain, which we postulated to be dispensable for catalytic activity, instead representing an isozyme-specific control domain within the overall architecture of NMNAT2. Indeed, we experimentally confirmed that removal of different-length fragments from this central domain did not compromise the enzyme's catalytic activity or the overall tridimensional structure of the active site.

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Different clinicians may do the same work in different ways, depending on their preferences and level of expertise. The nature and amount of clinical support required also varies with the knowledge level of the clinician: novice clinicians may require guidance at each and every stage of the process, while experienced clinicians like to have a free hand and need guidance only when in doubt. We describe a descriptive support system, where guidance is provided only when asked, rather than actively prescribing a list of actions at every stage of the clinical process. The system infers the state of the clinical pathway for a patient by examining the state of information in the Electronic Record that is created or modified during the patient treatment process; therefore, the clinician does not have to notify the system of every action he does while treating a patient, but the system can keep track of patient treatment so that appropriate guidance can be provided when needed.