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1.  Pyrazinoic Acid Efflux Rate in Mycobacterium Tuberculosis is a better proxy of Pyrazinamide Resistance 
Pyrazinamide is one of the most important drugs in the treatment of latent Mycobacterium tuberculosis infection. The emergence of strains resistant to pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include pyrazinamide. The accepted mechanism of action states that after the conversion of pyrazinamide into pyrazinoic acid by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. The pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of pyrazinamidase activity significantly contribute to pyrazinamide resistance, the mechanism of resistance is not completely understood.
The pyrazinoic acid efflux rate may depend on multiple factors, including pyrazinamidase activity, intracellular pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the pyrazinoic acid efflux rate to the susceptibility to pyrazinamide is recognized, its quantitative effect remains unknown.
Thirty-four M. tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which pyrazinoic acid is released from the bacteria and the initial velocity at which pyrazinamide enters the bacteria were estimated.
The data indicated that pyrazinoic acid efflux rates for pyrazinamide-susceptible M. tuberculosis strains fell within a specific range, and M. tuberculosis strains with a pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high pyrazinoic acid efflux rate for Mycobacterium smegmatis, which is innately resistant to pyrazinamide: its pyrazinoic acid efflux rate was found to be 900 fold higher than the average efflux rate for M. tuberculosis strains.
No significant variability was observed in the pyrazinamide flux rate. The pyrazinoic acid efflux rate explained 61% of the variability in Bactec pyrazinamide susceptibility, 24% of Wayne activity, and 51% of the Bactec 460TB growth index. In contrast, pyrazinamidase activity accounted for only 27% of the Bactec pyrazinamide susceptibility. This finding suggests that mechanisms other than pncA mutations (reduction of pyrazinamidase activity) are also implicated in pyrazinamide resistance, and that pyrazinoic acid efflux rate acts as a better proxy for pyrazinamide resistance than the presence of pncA mutations. This is relevant to the design of molecular diagnostics for pyrazinamide susceptibility, which currently rely on pncA gene mutation detection.
doi:10.1016/j.tube.2011.09.002
PMCID: PMC3269536  PMID: 22004792
Mycobacterium tuberculosis; POA efflux rate; PZA flux rate; PZA resistance
2.  Combined Whole-Cell High-Throughput Functional Screening for Identification of New Nicotinamidases/Pyrazinamidases in Metagenomic/Polygenomic Libraries 
Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide (NAM) to produce ammonia and nicotinic acid (NA). These enzymes are an essential component of the NAD+ salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or NA) to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside (SNP). After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the SNP method allowed the finding of the first nicotinamidase with balanced catalytic efficiency toward NAM (nicotinamidase activity) and pyrazinamide (pyrazinamidase activity). Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.
doi:10.3389/fmicb.2016.01915
PMCID: PMC5147024  PMID: 28018295
amidohydrolases; nicotinamidase; functional screening; metagenome; library; high-throughput screening assays
3.  Crystal Structure of the Pyrazinamidase of Mycobacterium tuberculosis: Insights into Natural and Acquired Resistance to Pyrazinamide 
PLoS ONE  2011;6(1):e15785.
Pyrazinamidase (PncA) activates the first-line antituberculous drug pyrazinamide into pyrazinoic acid. The crystal structure of the Mycobacterium tuberculosis PncA protein has been determined, showing significant differences in the substrate binding cavity when compared to the pyrazinamidases from Pyrococcus horikoshii and Acinetobacter baumanii. In M. tuberculosis, this region was found to hold a Fe2+ ion coordinated by one aspartate and three histidines, one of them corresponding to His57 which is replaced by Asp in Mycobacterium bovis, a species naturally resistant to pyrazinamide. The binding cavity also contains a Cys138-Asp8-Lys96 motif evocating a cysteine-based catalytic mechanism. Mutants have been constructed and investigated by kinetic and thermal shift assays, highlighting the importance of protein folding and thermal stability in the pyrazinamidase activity.
doi:10.1371/journal.pone.0015785
PMCID: PMC3025910  PMID: 21283666
4.  World-wide variation in incidence of Acinetobacter associated ventilator associated pneumonia: a meta-regression 
BMC Infectious Diseases  2016;16:577.
Background
Acinetobacter species such as Acinetobacter baumanii are of increasing concern in association with ventilator associated pneumonia (VAP). In the ICU, Acinetobacter infections are known to be subject to seasonal variation but the extent of geographic variation is unclear. The objective here is to define the extent and possible reasons for geographic variation for Acinetobacter associated VAP whether or not these isolates are reported as Acinetobacter baumanii.
Methods
A meta-regression model of VAP associated Acinetobacter incidence within the published literature was undertaken using random effects methods. This model incorporated group level factors such as proportion of trauma admissions, year of publication and reporting practices for Acinetobacter infection.
Results
The search identified 117 studies from seven worldwide regions over 29 years. There is significant variation in Acinetobacter species associated VAP incidence among seven world-wide regions. The highest incidence is amongst reports from the Middle East (mean; 95 % confidence interval; 8.8; 6 · 2–12 · 7 per 1000 mechanical ventilation days) versus that from North American ICU’s (1 · 2; 0 · 8–2 · 1). There is a similar geographic related disparity in incidence among studies reporting specifically as Acinetobacter baumanii. The incidence in ICU’s with a majority of admission being for trauma is >2.5 times that of other ICU’s.
Conclusion
There is greater than fivefold variation in Acinetobacter associated VAP among reports from various geographic regions worldwide. This variation is not explainable by variations in rates of VAP overall, admissions for trauma, publication year or Acinetobacter reporting practices as group level variables.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-016-1921-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12879-016-1921-4
PMCID: PMC5070388  PMID: 27756238
Intensive care unit; Geographic variation; Ventilator associated pneumonia; Acinetobacter
5.  Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA. 
Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.
PMCID: PMC163344  PMID: 8726014
6.  Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase from Mycobacterium smegmatis 
Journal of Bacteriology  1998;180(22):5809-5814.
The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable Km values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 μg/ml.
PMCID: PMC107651  PMID: 9811635
7.  Mortality Audit of Neonatal Sepsis Secondary to Acinetobacter 
Background:
Multidrug resistant Acinetobacter infection has emerged as an important pathogen in neonatal sepsis in the recent years causing morbidity as well as mortality.
Materials and Methods:
A retrospective analysis was performed over a one and a half year period of all neonates admitted with sepsis in our neonatal intensive care unit (NICU), who developed Acinetobacter infection and to identify mortality-associated risk factors in these neonates.
Results:
Incidence of neonatal septicaemia due to Acinetobacter species was 9.18%. All were cases of early onset sepsis. Predominant species isolated was Acinetobacter baumanii (67.5%). The major symptoms were lethargy and poor feeding. The major signs were tachypnoea, rib retraction, and respiratory distress. The major fetal risk factors were low birth weight and prematurity. Overall, 53.75% were multidrug resistant (MDR) Acinetobacter. Resistance to more than two drugs (MDR) was statistically significant in A. baumanii as compared with nonbaumanii. Overall mortality due to Acinetobacter neonatal sepsis was 20%. Septicemia due to A. baumanii was associated with higher mortality than those due to nonbaumanii isolates. Lethargy, tachypnoea, rib retraction, tachycardia, respiratory distress, and mechanical ventilation were significant predictors of mortality.
Conclusion:
Multidrug resistant Acinetobacter infection is fatal, particularly in premature and low birth weight neonates. Therefore, an effective infection control policy and rational antibiotic use are mandatory in neonatal intensive care areas of each hospital in order to control Acinetobacter infection and improve outcome.
doi:10.4103/0974-777X.107165
PMCID: PMC3628231  PMID: 23599610
Acinetobacter sepsis; Mortality; Neonates
8.  Reduced Pyrazinamidase Activity and the Natural Resistance of Mycobacterium kansasii to the Antituberculosis Drug Pyrazinamide 
Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug that requires conversion to the bactericidal compound pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) activity of nicotinamidase to show activity against Mycobacterium tuberculosis. Mutations leading to a loss of PZase activity cause PZA resistance in M. tuberculosis. M. kansasii is naturally resistant to PZA and has reduced PZase activity along with an apparently detectable nicotinamidase activity. The role of the reduction in PZase activity in the natural PZA resistance of M. kansasii is unknown. The MICs of PZA and POA for M. kansasii were determined to be 500 and 125 μg/ml, respectively. Using [14C]PZA and [14C]nicotinamide, we found that M. kansasii had about 5-fold-less PZase activity and about 25-fold-less nicotinamidase activity than M. tuberculosis. The M. kansasii pncA gene was cloned on a 1.8-kb BamHI DNA fragment, using M. avium pncA probe. Sequence analysis showed that the M. kansasii pncA gene encoded a protein with homology to its counterparts from M. tuberculosis (69.9%), M. avium (65.6%), and Escherichia coli (28.5%). Transformation of naturally PZA-resistant M. bovis BCG with M. kansasii pncA conferred partial PZA susceptibility. Transformation of M. kansasii with M. avium pncA caused functional expression of PZase and high-level susceptibility to PZA, indicating that the natural PZA resistance in M. kansasii results from a reduced PZase activity. Like M. tuberculosis, M. kansasii accumulated POA in the cells at an acidic pH; however, due to its highly active POA efflux pump, the naturally PZA-resistant species M. smegmatis did not. These findings suggest the existence of a weak POA efflux mechanism in M. kansasii.
PMCID: PMC89157  PMID: 10049264
9.  Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831 
PLoS ONE  2013;8(2):e56727.
Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD+ salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (kcat/Km = 43.5 mM−1s−1) than pyrazinamidase (kcat/Km = 3.2 mM−1s−1). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.
doi:10.1371/journal.pone.0056727
PMCID: PMC3581539  PMID: 23451075
10.  Revisiting Anti-tuberculosis Activity of Pyrazinamide in Mice 
The mechanism of action of pyrazinamide, a key sterilizing drug in the treatment of tuberculosis, remains elusive; pyrazinamide is a pro-drug that requires activation by a bacterial-encoded enzyme, and its activity is most apparent on non-replicating Mycobacterium tuberculosis. Recently, it has been suggested that pyrazinamide might exert also some host-directed effect in addition to its antimicrobial activity. To address this possibility, three sequential experiments were conducted in immune-competent BALB/c and in immune-deficient, athymic nude mice. In the first experiment, BALB/c mice infected with M. bovis, which is naturally resistant to pyrazinamide because it is unable to activate the drug, were treated with standard drug regimens with and without pyrazinamide to specifically detect a host-directed effect. As no effect was observed, pyrazinamide activity was compared in M. tuberculosis-infected BALB/c and nude mice to determine whether the effect of pyrazinamide would be reduced in the immune deficient mice. As pyrazinamide did not appear to have any affect in the nude mice, a third experiment was performed in which rifampin was replaced with rifapentine (a similar drug with a longer half-life) to permanently suppress mycobacterial growth. In these experimental conditions, the antimicrobial effect of pyrazinamide was clear. Therefore, the results of our studies rule out a significant host-directed effect of pyrazinamide in the TB infected host.
doi:10.4172/2161-1068.1000145
PMCID: PMC4267256  PMID: 25525563
11.  Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis 
Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.
doi:10.1038/emi.2014.61
PMCID: PMC4150287  PMID: 26038753
Pyrazinamide; pyrazinoic acid resistance; mode of action; panD; aspartate decarboxylase
12.  Kinetics and Inhibition of Nicotinamidase from Mycobacterium tuberculosis 
Biochemistry  2010;49(44):9613-9619.
Nicotinamidase/Pyrazinamidase (PncA) is involved in the NAD+ salvage pathway of Mycobacterium tuberculosis and other bacteria. In addition to hydrolyzing nicotinamide into nicotinic acid, PncA also hydrolyzes the pro-drug pyrazinamide to generate the active form of the drug, pyrazinoic acid, which is an essential component of the multidrug treatment of TB. A coupled enzymatic activity assay has been developed for PncA that allows for the spectroscopic observation of enzyme activity. The enzyme activity was essentially pH independent under the conditions tested, however, the measurement of the pH dependence of iodoacetamide alkylation revealed a pK value of 6.6 for the active site cysteine. Solvent deuterium kinetic isotope effects revealed an inverse value on kcat of 0.64, reconfirming the involvement of a thiol group in the mechanism. A mechanism is proposed for PncA catalysis that is similar to the mechanisms proposed for members of the Nitrilase superfamily, in which nucleophilic attack by the active site cysteine generates a tetrahedral intermediate that collapses with the loss of ammonia and subsequent hydrolysis of the thioester bond by water completes the cycle. An inhibitor screen identified the competitive inhibitor 3-pyridine carboxaldehyde with a Ki of 290 nM. Additionally, pyrazinecarbonitrile was found to be an irreversible inactivator of PncA, with a kinact/KI of 975 M−1sec−1.
doi:10.1021/bi1011157
PMCID: PMC3029496  PMID: 20879713
Pyrazinamide; Nicotinamidase; Pyrazinamidase; PncA; tuberculosis
13.  Mutations in panD encoding aspartate decarboxylase are associated with pyrazinamide resistance in Mycobacterium tuberculosis 
Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug susceptible and multidrug-resistant tuberculosis (MDR-TB). Resistance to PZA is most commonly associated with mutations in the pncA gene encoding nicotinamidase/pyrazinamidase which converts the prodrug PZA to the active form pyrazinoic acid (POA). RpsA (ribosomal protein S1) involved in trans-translation was recently shown to be a target of PZA and mutations in RpsA are found in some PZA-resistant TB strains. However, some other PZA-resistant strains lack mutations in either pncA or rpsA. To identify potential new mechanisms of PZA resistance, we isolated 174 in vitro mutants of M. tuberculosis H37Rv resistant to PZA to search for resistant isolates that do not have pncA or rpsA mutations. DNA sequencing revealed that 169 of the 174 (97.1%) PZA-resistant mutants had pncA mutations but 5 mutants lacked pncA or rpsA mutations. Whole genome sequencing analyses revealed that the 5 PZA-resistant mutants had different mutations all occurring in the same gene panD encoding aspartate decarboxylase, which is involved in synthesis of β-alanine that is a precursor for pantothenate and co-enzyme A biosynthesis. panD mutations were identified in naturally PZA-resistant Mycobacterium canetti strain and a PZA-resistant MDR-TB clinical isolate. Future studies are needed to address the role of panD mutations in PZA resistance and confirm PanD as a new target of PZA.
doi:10.1038/emi.2013.38
PMCID: PMC3697303  PMID: 26038471
aspartate decarboxylase; mechanism of resistance; mode of action; panD; pyrazinamide
14.  Monoclonal antibodies against the iron regulated outer membrane Proteins of Acinetobacter baumannii are bactericidal 
BMC Microbiology  2001;1:16.
Background
Iron is an important nutrient required by all forms of life.In the case of human hosts,the free iron availability is 10-18M,which is far less than what is needed for the survival of the invading bacterial pathogen.To survive in such conditions, bacteria express new proteins in their outer membrane and also secrete iron chelators called siderophores.
Results/ Discussion
Acinetobacter baumannii ATCC 19606, a nosocomial pathogen which grows under iron restricted conditions, expresses four new outer membrane proteins,with molecular weight ranging from 77 kDa to 88 kDa, that are called Iron Regulated Outer Membrane Proteins (IROMPs). We studied the functional and immunological properties of IROMPs expressed by A.baumanii ATCC 19606.The bands corresponding to IROMPs were eluted from SDS-PAGE and were used to immunize BALB/c mice for the production of monoclonal antibodies. Hybridomas secreting specific antibodies against these IROMPs were selected after screening by ELISA and their reactivity was confirmed by Western Blot. The antibodies then generated belonged to IgM isotype and showed bactericidical and opsonising activities against A.baumanii in vitro.These antibodies also blocked siderophore mediated iron uptake via IROMPs in bacteria.
Conclusion
This proves that iron uptake via IROMPs,which is mediated through siderophores,may have an important role in the survival of A.baumanii inside the host,and helps establishing the infection.
PMCID: PMC48144  PMID: 11532195
15.  Enhanced Tolerance against Early and Late Apoptotic Oxidative Stress in Mammalian Neurons through Nicotinamidase and Sirtuin Mediated Pathways 
Current neurovascular research  2008;5(3):159-170.
Focus upon therapeutic strategies that intersect between pathways that govern cellular metabolism and cellular survival may offer the greatest impact for the treatment of a number of neurodegenerative and metabolic disorders, such as diabetes mellitus. In this regard, we investigated the role of a Drosophila nicotinamidase (DN) in mammalian SH-SY5Y neuronal cells during oxidative stress. We demonstrate that during free radical exposure to nitric oxide generators DN neuronal expression significantly increased cell survival and blocked cellular membrane injury. Furthermore, DN neuronal expression prevented both apoptotic late DNA degradation and early phosphatidylserine exposure that may serve to modulate inflammatory cell activation in vivo. Nicotinamidase activity that limited nicotinamide cellular concentrations appeared to be necessary for DN neuroprotection, since application of progressive nicotinamide concentrations could abrogate the benefits of DN expression during oxidative stress. Pathways that involved sirtuin activation and SIRT1 were suggested to be vital, at least in part, for DN to confer protection through a series of studies. First, application of resveratrol increased cell survival during oxidative stress either alone or in conjunction with the expression of DN to a similar degree, suggesting that DN may rely upon SIRT1 activation to foster neuronal protection. Second, the overexpression of either SIRT1 or DN in neurons prevented apoptotic injury specifically in neurons expressing these proteins during oxidative stress, advancing the premise that DN and SIRT1 may employ similar pathways for neuronal protection. Third, inhibition of sirtuin activity with sirtinol was detrimental to neuronal survival during oxidative stress and prevented neuronal protection during overexpression of DN or SIRT1, further supporting that SIRT1 activity may be necessary for DN neuroprotection during oxidative stress. Implementation of further work to elucidate the cellular mechanisms that govern nicotinamidase activity in mammalian cells may offer novel avenues for the treatment of disorders tied to oxidative stress and cellular metabolic dysfunction.
PMCID: PMC2615543  PMID: 18691073
Apoptosis; neuron; nicotinamidase; oxidative stress; phosphatidylserine; resveratrol; sirtinol; sirtuin; SIRT1
16.  Clinical and antimicrobial profile of Acinetobacter spp.: An emerging nosocomial superbug 
Background:
Recently, Acinetobacter has emerged as significant hospital pathogen, notoriously known to acquire antibiotic resistance to most of the commonly prescribed antimicrobials. Many risk factors are associated with Acinetobacter infections, especially in patients in intensive care unit (ICU). This study aims to isolate Acinetobacter from various clinical specimens and to determine its antimicrobial sensitivity pattern.
Materials and Methods:
Identification, speciation and antimicrobial sensitivity testing were performed using the standard microbiological techniques. Slime production was also tested by microtiter plate and tube method.
Results:
From the processed clinical specimens, 107 Acinetobacter strains (1.02%) were isolated of which 76 (0.74%) isolates were from general wards and 31 (11.96%) were from ICU. Significantly higher percentage of Acinetobacter strains was found in ICU compared with general wards (P < 0.05). Most common Acinetobacter infection was abscess. Infections were more common in males and were associated with major risk factors such as post-surgical, diabetes mellitus, catheterization, extended hospital stay and prolonged antibiotic usage. Acinetobacter baumanii was the most common species isolated to cause abscess, wound infection, etc. 62.61% and 28.97% isolates produced slime by microtiter plate and tube method. Imipenem was most sensitive drug followed by amikacin. Ceftazidime, cefotaxime, piperacillin were most resistant. 43.00% isolates were IPM resistant. A. baumanii was more resistant to commonly used antimicrobials.
Conclusion:
Acinetobacter nosocomial infections resistant to most antimicrobials have emerged, especially in ICU. Early identification and continued surveillance of prevalent organism will help prevent the spread of Acinetobacter in hospital environment.
doi:10.4103/2277-9175.124642
PMCID: PMC3929011  PMID: 24600597
Acinetobacter; antimicrobial resistance; nosocomial pathogen
17.  The Human Milk Protein-Lipid Complex HAMLET Sensitizes Bacterial Pathogens to Traditional Antimicrobial Agents 
PLoS ONE  2012;7(8):e43514.
The fight against antibiotic resistance is one of the most significant challenges to public health of our time. The inevitable development of resistance following the introduction of novel antibiotics has led to an urgent need for the development of new antibacterial drugs with new mechanisms of action that are not susceptible to existing resistance mechanisms. One such compound is HAMLET, a natural complex from human milk that kills Streptococcus pneumoniae (the pneumococcus) using a mechanism different from common antibiotics and is immune to resistance-development. In this study we show that sublethal concentrations of HAMLET potentiate the effect of common antibiotics (penicillins, macrolides, and aminoglycosides) against pneumococci. Using MIC assays and short-time killing assays we dramatically reduced the concentrations of antibiotics needed to kill pneumococci, especially for antibiotic-resistant strains that in the presence of HAMLET fell into the clinically sensitive range. Using a biofilm model in vitro and nasopharyngeal colonization in vivo, a combination of HAMLET and antibiotics completely eradicated both biofilms and colonization in mice of both antibiotic-sensitive and resistant strains, something each agent alone was unable to do. HAMLET-potentiation of antibiotics was partially due to increased accessibility of antibiotics to the bacteria, but relied more on calcium import and kinase activation, the same activation pathway HAMLET uses when killing pneumococci by itself. Finally, the sensitizing effect was not confined to species sensitive to HAMLET. The HAMLET-resistant respiratory species Acinetobacter baumanii and Moraxella catarrhalis were all sensitized to various classes of antibiotics in the presence of HAMLET, activating the same mechanism as in pneumococci. Combined these results suggest the presence of a conserved HAMLET-activated pathway that circumvents antibiotic resistance in bacteria. The ability to activate this pathway may extend the lifetime of the current treatment arsenal.
doi:10.1371/journal.pone.0043514
PMCID: PMC3419703  PMID: 22905269
18.  Daily Dosing of Rifapentine Cures Tuberculosis in Three Months or Less in the Murine Model 
PLoS Medicine  2007;4(12):e344.
Background
Availability of an ultra-short-course drug regimen capable of curing patients with tuberculosis in 2 to 3 mo would significantly improve global control efforts. Because immediate prospects for novel treatment-shortening drugs remain uncertain, we examined whether better use of existing drugs could shorten the duration of treatment. Rifapentine is a long-lived rifamycin derivative currently recommended only in once-weekly continuation-phase regimens. Moxifloxacin is an 8-methoxyfluoroquinolone currently used in second-line regimens.
Methods and Findings
Using a well-established mouse model with a high bacterial burden and human-equivalent drug dosing, we compared the efficacy of rifapentine- and moxifloxacin-containing regimens with that of the standard daily short-course regimen based on rifampin, isoniazid, and pyrazinamide. Bactericidal activity was assessed by lung colony-forming unit counts, and sterilizing activity was assessed by the proportion of mice with culture-positive relapse after 2, 3, 4, and 6 mo of treatment. Here, we demonstrate that replacing rifampin with rifapentine and isoniazid with moxifloxacin dramatically increased the activity of the standard daily regimen. After just 2 mo of treatment, mice receiving rifapentine- and moxifloxacin-containing regimens were found to have negative lung cultures, while those given the standard regimen still harbored 3.17 log10 colony-forming units in the lungs (p < 0.01). No relapse was observed after just 3 mo of treatment with daily and thrice-weekly administered rifapentine- and moxifloxacin-containing regimens, whereas the standard daily regimen required 6 mo to prevent relapse in all mice.
Conclusions
Rifapentine should no longer be viewed solely as a rifamycin for once-weekly administration. Our results suggest that treatment regimens based on daily and thrice-weekly administration of rifapentine and moxifloxacin may permit shortening the current 6 mo duration of treatment to 3 mo or less. Such regimens warrant urgent clinical investigation.
Eric Nuermberger and colleagues found that after two months of treatment, mice with lung cultures positive for tuberculosis that received daily doses of rifapentine- and moxifloxacin-containing regimens converted to negative lung cultures. This finding could make possible the development of shorter treatment regimens for humans.
Editors' Summary
Background.
Every year, nearly 9 million people develop tuberculosis—a bacterial infection most commonly of the lungs—and about 2 million people die from the disease. Tuberculosis is caused by Mycobacterium tuberculosis, bacteria that are spread in airborne droplets when people with active tuberculosis sneeze or cough. Most infected people never become ill—their immune system successfully contains the infection. However, the bacteria remain dormant within the body and can cause disease years later if host immunity declines. Active tuberculosis can be cured by taking several antibiotics daily (for tuberculosis treatments, daily may mean five or seven times a week) for at least 6 mo. Combinations of drugs are needed to prevent the bacteria from developing resistance to the treatment, but also because of the complex biology of M. tuberculosis. During active tuberculosis, there are rapidly multiplying bacteria in the lungs but also less rapidly multiplying and near-dormant bacteria elsewhere in the body. Effective treatments contain a “bactericidal” drug such as isoniazid to kill the actively multiplying bacteria, a drug to kill the less actively multiplying bugs (for example, pyrazinamide), and a sterilizing drug (the most potent of which is rifampin) to kill the near-dormant bacteria and thus prevent the disease from recurring.
Why Was This Study Done?
Unfortunately, many patients fail to complete this treatment because it is long and complicated and because the drugs may have unpleasant side effects. Poor adherence to treatment contributes to the emergence of drug resistance and means that people stay infectious for longer and are more likely to have relapses. Consequently, it is hampering global efforts to control tuberculosis. A shorter course of treatment might improve matters, but many researchers believe that this will require the development of new drugs and, although there are several promising candidates, it will be several years before they can be used in patients. In this study, therefore, the researchers asked whether better use of existing drugs could shorten treatment times. In particular, they studied tuberculosis in animals to investigate whether a long-lived rifampin-like drug called rifapentine combined with moxifloxacin (an alternative to isoniazid) might shorten treatment times.
What Did the Researchers Do and Find?
The researchers used several different courses (“regimens”) of treatment containing rifapentine, moxifloxacin, and pyrazinamide, and the standard daily short-course regimen containing rifampin, isoniazid, and pyrazinamide to treat mice infected with M. tuberculosis. For each regimen, they measured its bactericidal activity by counting how many bacterial colonies could be grown from the lungs of the mice at specific times during the treatment, and its sterilizing activity by assessing the proportion of mice with any live bacteria in their lungs (a culture-positive relapse) after treatment completion. After 2 mo of treatment, the mice receiving the rifapentine- and moxifloxacin-containing regimens had negative lung cultures, a point not reached with the standard regimen until after 4 mo of treatment. Three months of treatment with daily or thrice-weekly rifapentine- and moxifloxacin-containing regimens was sufficient to prevent any culture-positive relapses, whereas the standard daily regimen had to be continued for 6 mo to achieve cure. Testing of additional drug combinations revealed that rifapentine is the most important drug in the new regimen and that simply replacing rifampin with rifapentine and retaining isoniazid also is sufficient to shorten the duration of therapy to 3 mo in this experimental model.
What Do These Findings Mean?
These findings provide the first evidence that replacing rifampin with rifapentine might halve the length of therapy needed to cure tuberculosis. They also indicate that it might be possible to give the drugs thrice-weekly rather than daily as in the current therapy. The World Health Organization recommends that all tuberculosis treatment is supervised (so-called directly observed therapy) to ensure treatment adherence, so a regimen that requires only three doses a week for 3 mo would greatly reduce the resources needed to treat tuberculosis as well as potentially improving treatment adherence. However, it should be emphasized that the current study is experimental, and there may be important differences between how mice and people respond to the proposed drug regimens, both in terms of cure rates and side effects. Nevertheless, these results strongly suggest that the safety, tolerability, and efficacy of tuberculosis treatment regimens containing rifapentine and pyrazinamide, combined with either moxifloxacin or isoniazid, should be evaluated in people as soon as possible.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040344.
The MedlinePlus encyclopedia contains a page on tuberculosis (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases provides information on all aspects of tuberculosis
The US Centers for Disease Control and Prevention provide several fact sheets and other information resources about tuberculosis, including information for patients and caregivers about treatment adherence
The World Health Organization provides a 2007 report on global tuberculosis control (in English with key findings in French and Spanish), information on the Stop TB initiative, and a recent bulletin on tuberculosis treatment (in English with an abstract in French, Spanish, and Arabic)
doi:10.1371/journal.pmed.0040344
PMCID: PMC2140085  PMID: 18092886
19.  STRUCTURES OF THE CLASS D CARBAPENEMASE OXA-24 FROM ACINETOBACTER BAUMANNII IN COMPLEX WITH DORIPENEM 
Journal of molecular biology  2011;406(4):583-594.
The emergence of class D β-lactamases with carbapenemase activity presents an enormous challenge to health practitioners, particularly with regard to the treatment of infections caused by Gram negative pathogens such as Acinetobacter baumanii. Unfortunately, class D β-lactamases with carbapenemase activity are resistant to β-lactamase inhibitors. To better understand the details of the how these enzymes bind and hydrolyze carbapenems, we have determined the structures of two deacylation-deficient variants (K84D and V130D) of the class D carbapenemase OXA-24 with doripenem bound as a covalent acyl-enzyme intermediate. Doripenem adopts essentially the same configuration in both OXA-24 variant structures, but varies significantly when compared to the non-carbapenemase class D member OXA-1/doripenem complex. The alcohol of the 6α hydroxyethyl moiety is directed away from the general base carboxy-K84, with implications for activation of the deacylating water. The tunnel formed by the Y112/M223 bridge in the apo form of OXA-24 is largely unchanged by the binding of doripenem. The presence of this bridge, however, causes the distal pyrrolidine/sulfonamide group to bind in a drastically different conformation compared to doripenem bound to OXA-1. The resulting difference in the position of the side-chain bridge sulfur of doripenem is consistent with the hypothesis that the tautomeric state of the pyrroline ring contributes to the different carbapenem hydrolysis rates of OXA-1 and OXA-24. These findings represent a snapshot of a key step in the catalytic mechanism of an important class D enzyme, and may be useful for the design of novel inhibitors.
doi:10.1016/j.jmb.2010.12.042
PMCID: PMC3057435  PMID: 21215758
class D β-lactamase; acyl-enzyme; carbapenem; deacylation-deficient; tautomerization
20.  Enhanced Bactericidal Activity of Rifampin and/or Pyrazinamide When Combined with PA-824 in a Murine Model of Tuberculosis ▿  
Antimicrobial Agents and Chemotherapy  2008;52(10):3664-3668.
PA-824 is in phase II clinical testing to treat tuberculosis. At a dose of 100 mg/kg of body weight, it has demonstrated bactericidal activity during the initial and continuation phases of treatment in a murine model of tuberculosis. In a prior study, substitution of PA-824 for isoniazid in the first-line regimen of rifampin, isoniazid, and pyrazinamide resulted in significantly lower CFU counts at 2 months and shorter time to culture-negative conversion. However, the study design prevented a rigorous assessment of the relapse rate after completion of therapy. The current experiment was designed to assess (i) the extent of the beneficial effect of substituting PA-824 for isoniazid in the first-line regimen, (ii) the influence of the PA-824 dose on the same effect, and (iii) the activity of each one-, two-, and three-drug combination of rifampin, PA-824, and pyrazinamide. Mice were infected by the aerosol route and initiated on treatment 14 days later with more than 7 log10 CFU per lung. Treatment with rifampin and pyrazinamide was more effective than treatment with rifampin, isoniazid, and pyrazinamide at reducing the lung CFU count, consistent with past evidence of isoniazid's antagonism in this model. The addition of PA-824 at 12.5 and 25 mg/kg/day did not increase the activity of rifampin plus pyrazinamide, but the addition of PA-824 at 50 and 100 mg/kg/day did increase the activity in a dose-dependent manner. The combination of rifampin, PA-824 (100 mg/kg), and pyrazinamide rendered all mice culture negative after 2 months of treatment and free of relapse after 4 months of treatment, while some mice receiving rifampin, isoniazid, and pyrazinamide remained culture positive and 15% relapsed after completing 4 months of treatment. The two-drug combination of PA-824 and pyrazinamide displayed synergistic activity that was equivalent to that of the standard first-line regimen. Together, these results support the evaluation of regimens based on the combination of rifampin, PA-824, and pyrazinamide in phase II clinical trials while demonstrating several potential pitfalls in the evaluation of new drug combinations in a murine model of tuberculosis.
doi:10.1128/AAC.00686-08
PMCID: PMC2565869  PMID: 18694943
21.  Coexistence of Extended Spectrum Beta-Lactamases, AmpC Beta-Lactamases and Metallo-Beta-Lactamases in Acinetobacter baumannii from burns patients: a report from a tertiary care centre of India 
Summary
Multidrug-resistant Acinetobacter baumanii is a major pathogen encountered in pyogenic infections, especially from burns patients in hospital settings. Often there is also coexistence of multiple beta-lactamase enzymes responsible for beta-lactam resistance in a single isolate, which further complicates treatment options. We conducted a study on burn wound pus samples obtained from the burns unit of our hospital. Phenotypic tests were used to determine the Extended Spectrum Beta-Lactamase, AmpC Beta-Lactamase and Metallo-Beta-Lactamase producing status of the isolates. Almost half of the samples from the burn wounds yielded Acinetobacter baumanii as the predominant pathogen (54.05%). Coexistence of the three resistance mechanisms was seen in 25 of the 100 (25%) isolates of Acinetobacter baumanii. This study emphasizes the need for the detection of isolates that produce these enzymes to avoid therapeutic failures and nosocomial outbreaks.
PMCID: PMC3978590  PMID: 24799848
Acinetobacter baumanii; Extended Spectrum Beta-Lactamase; AmpC Beta-Lactamase; Metallo-Beta-Lactamase
22.  Diversity, evolution and medical applications of insect antimicrobial peptides 
Antimicrobial peptides (AMPs) are short proteins with antimicrobial activity. A large portion of known AMPs originate from insects, and the number and diversity of these molecules in different species varies considerably. Insect AMPs represent a potential source of alternative antibiotics to address the limitation of current antibiotics, which has been caused by the emergence and spread of multidrug-resistant pathogens. To get more insight into AMPs, we investigated the diversity and evolution of insect AMPs by mapping their phylogenetic distribution, allowing us to predict the evolutionary origins of selected AMP families and to identify evolutionarily conserved and taxon-specific families. Furthermore, we highlight the use of the nematode Caenorhabditis elegans as a whole-animal model in high-throughput screening methods to identify AMPs with efficacy against human pathogens, including Acinetobacter baumanii and methicillin-resistant Staphylococcus aureus. We also discuss the potential medical applications of AMPs, including their use as alternatives for conventional antibiotics in ectopic therapies, their combined use with antibiotics to restore the susceptibility of multidrug-resistant pathogens, and their use as templates for the rational design of peptidomimetic drugs that overcome the disadvantages of therapeutic peptides.
The article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’.
doi:10.1098/rstb.2015.0290
PMCID: PMC4874388  PMID: 27160593
antimicrobial peptides; anti-infective; drug development; evolution; immunity; insects
23.  Proceedings of the 15th Annual UT-KBRIN Bioinformatics Summit 2016 
Rouchka, Eric C. | Chariker, Julia H. | Harrison, Benjamin J. | Park, Juw Won | Cao, Xueyuan | Pounds, Stanley | Raimondi, Susana | Downing, James | Ribeiro, Raul | Rubnitz, Jeffery | Lamba, Jatinder | Daigle, Bernie J. | Burgess, Deborah | Gehrlich, Stephanie | Carmen, John C. | Johnson, Nicholas | Emani, Chandrakanth | Gehrlich, Stephanie | Burgess, Deborah | Carmen, John C. | De Silva, Kalpani | Heaton, Michael P. | Kalbfleisch, Theodore S. | Viangteeravat, Teeradache | Mudunuri, Rahul | Ajayi, Oluwaseun | Şen, Fatih | Huang, Eunice Y. | Mohebbi, Mohammad | Florian, Luaire | Jackson, Douglas J. | Naber, John F. | Sabbir, AKM | Ellingson, Sally R. | Lu, Yuping | Phillips, Charles A | Langston, Michael A. | Sevakula, Rahul K. | Thirukovalluru, Raghuveer | Verma, Nishchal K. | Cui, Yan | Sayed, Mohammed | Park, Juw Won | Wang, Jing | Liu, Qi | Shyr, Yu | Zhang, Xiaofei | Ellingson, Sally R. | Prodduturi, Naresh | Oliver, Gavin R. | Grill, Diane | Na, Jie | Eckel-Passow, Jeanette | Klee, Eric W. | Goodin, Michael M. | Farman, Mark | Inocencio, Harrison | Jang, Chanyong | Jaromczyk, Jerzy W. | Moore, Neil | Sovacool, Kelly | Dent, Leon | Izban, Mike | Mandape, Sammed | Sakhare, Shruti | Pratap, Siddharth | Marshall, Dana | DePriest, M Scotty | MacLeod, James N. | Kalbfleisch, Theodore S. | Emani, Chandrakanth | Adam, Hanady | Blandford, Ethan | Campbell, Joel | Castlen, Joshua | Dixon, Brittany | Gilbert, Ginger | Hall, Aaron | Kreisle, Philip | Lasher, Jessica | Oakes, Bethany | Speer, Allison | Valentine, Maximilian | Nagisetty, Naga Satya V. Rao | Jose, Rony | Viangteeravat, Teeradache | Rooney, Robert | Hains, David
BMC Bioinformatics  2016;17(Suppl 10):297.
Table of contents
I1 Proceedings of the Fifteenth Annual UT- KBRIN Bioinformatics Summit 2016
Eric C. Rouchka, Julia H. Chariker, Benjamin J. Harrison, Juw Won Park
P1 CC-PROMISE: Projection onto the Most Interesting Statistical Evidence (PROMISE) with Canonical Correlation to integrate gene expression and methylation data with multiple pharmacologic and clinical endpoints
Xueyuan Cao, Stanley Pounds, Susana Raimondi, James Downing, Raul Ribeiro, Jeffery Rubnitz, Jatinder Lamba
P2 Integration of microRNA-mRNA interaction networks with gene expression data to increase experimental power
Bernie J Daigle, Jr.
P3 Designing and writing software for in silico subtractive hybridization of large eukaryotic genomes
Deborah Burgess, Stephanie Gehrlich, John C Carmen
P4 Tracking the molecular evolution of Pax gene
Nicholas Johnson; Chandrakanth Emani
P5 Identifying genetic differences in thermally dimorphic and state specific fungi using in silico genomic comparison
Stephanie Gehrlich, Deborah Burgess, John C Carmen
P6 Identification of conserved genomic regions and variation therein amongst Cetartiodactyla species using next generation sequencing
Kalpani De Silva, Michael P Heaton, Theodore S Kalbfleisch
P7 Mining physiological data to identify patients with similar medical events and phenotypes
Teeradache Viangteeravat, Rahul Mudunuri, Oluwaseun Ajayi, Fatih Şen, Eunice Y Huang
P8 Smart brief for home health monitoring
Mohammad Mohebbi, Luaire Florian, Douglas J Jackson, John F Naber
P9 Side-effect term matching for computational adverse drug reaction predictions
AKM Sabbir, Sally R Ellingson
P10 Enrichment vs robustness: A comparison of transcriptomic data clustering metrics
Yuping Lu, Charles A Phillips, Michael A Langston
P11 Deep neural networks for transcriptome-based cancer classification
Rahul K Sevakula, Raghuveer Thirukovalluru, Nishchal K. Verma, Yan Cui
P12 Motif discovery using K-means clustering
Mohammed Sayed, Juw Won Park
P13 Large scale discovery of active enhancers from nascent RNA sequencing
Jing Wang, Qi Liu, Yu Shyr
P14 Computationally characterizing genomic pipelines and benchmarking results using GATK best practices on the high performance computing cluster at the University of Kentucky
Xiaofei Zhang, Sally R Ellingson
P15 Development of approaches enabling the identification of abnormal gene expression from RNA-Seq in personalized oncology
Naresh Prodduturi, Gavin R Oliver, Diane Grill, Jie Na, Jeanette Eckel-Passow, Eric W Klee
P16 Processing RNA-Seq data of plants infected with coffee ringspot virus
Michael M Goodin, Mark Farman, Harrison Inocencio, Chanyong Jang, Jerzy W Jaromczyk, Neil Moore, Kelly Sovacool
P17 Comparative transcriptomics of three Acinetobacter baumanii clinical isolates with different antibiotic resistance patterns
Leon Dent, Mike Izban, Sammed Mandape, Shruti Sakhare, Siddharth Pratap, Dana Marshall
P18 Metagenomic assessment of possible microbial contamination in the equine reference genome assembly
M Scotty DePriest, James N MacLeod, Theodore S Kalbfleisch
P19 Molecular evolution of cancer driver genes
Chandrakanth Emani, Hanady Adam, Ethan Blandford, Joel Campbell, Joshua Castlen, Brittany Dixon, Ginger Gilbert, Aaron Hall, Philip Kreisle, Jessica Lasher, Bethany Oakes, Allison Speer, Maximilian Valentine
P20 Biorepository Laboratory Information Management System
Naga Satya V Rao Nagisetty, Rony Jose, Teeradache Viangteeravat, Robert Rooney, David Hains
doi:10.1186/s12859-016-1154-y
PMCID: PMC5001208
24.  High Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into Catalytic Mechanism and Inhibition by Aldehydes∥,‡ 
Biochemistry  2010;49(40):8803-8812.
Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD+ in most prokaryotes, most single cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD+ homeostasis has increased interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD+ consuming enzymes, such as the NAD+-dependent deacetylases (sirtuins). Here, we report several high resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. The structure of the C136S mutant in complex with nicotinamide provides details about substrate binding while a trapped nicotinoyl-thioester complexed with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features including a metal ion that coordinates the substrate and the catalytically relevant water molecule, and an oxyanion hole which both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence for several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.
doi:10.1021/bi1012436
PMCID: PMC3006156  PMID: 20853856
25.  Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from S. cerevisiae† 
Biochemistry  2011;51(1):243-256.
Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD+ salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from S. cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction where ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogs acting as inhibitors or substrates were examined revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary 15N and 13C kinetic isotope effects (KIE) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was observed indicative of a higher commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism is discussed involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid. These results will aid design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.
doi:10.1021/bi2015508
PMCID: PMC3257521  PMID: 22229411

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