Characterization of a transient covalent adduct formed during dimethylarginine dimethylaminohydrolase catalysis

Dimethylarginine dimethylaminohydrolase (DDAH) regulates the concentrations of human endogenous inhibitors of nitric oxide synthase, N(omega)-methyl-l-arginine (NMMA), and asymmetric N(omega),N(omega)-dimethyl-l-arginine (ADMA). Pharmacological regulation of nitric oxide synthesis is an important goal, but the catalytic mechanism of DDAH remains largely unexplored. A DDAH from Pseudomonas aeruginosa was cloned, and asymmetrically methylated arginine analogues were shown to be the preferred substrates, with ADMA displaying a slightly higher k(cat)/K(M) value than NMMA. DDAH is similar to members of a larger superfamily of guanidino-modifying enzymes, some of which have been shown to use an S-alkylthiouronium intermediate during catalysis. No covalent intermediates were found to accumulate during steady-state turnover reactions of DDAH with NMMA or ADMA. However, identification of a new substrate with an activated leaving group, S-methyl-l-thiocitrulline (SMTC), enabled acid trapping and ESI-MS characterization of a transient covalent adduct with a mass of 158 +/- 10 Da that accumulates during steady-state turnover. Subsequent trapping, proteolysis, peptide mapping and fragmentation by mass spectrometry, and site-directed mutagenesis demonstrated that this covalent adduct was attached to an active site residue and implicates Cys249 as the catalytic nucleophile required for intermediate formation. The use of covalent catalysis clearly links DDAH to this superfamily of enzymes and suggests that an S-alkylthiouronium intermediate may be a conserved feature in their mechanisms.     

Metallo-β-lactamase: Inhibitors and reporter substrates

Metallo-β-lactamases represent an emerging clinical threat due to their ability to render ineffective an entire class of antibiotics. Accordingly, this family of enzymes has been suggested as an attractive target for drug design. Progress toward developing effective inhibitors as well as the development of reporter substrates is reviewed. Inhibitors are classified into six classes and known binding interactions with metallo-β-lactamases are summarized. The development of chromogenic and fluorogenic reporter substrates is also reviewed with respect to current and prospective applications to future inhibitor and diagnostic discovery, mechanistic studies, and biological imaging. Despite progress in molecular probe development, the sequence and structural diversity within the metallo-β-lactamase family continue to present substantial hurdles for rational ligand design.     

HIV-specific CD8+ T cell function in children with vertically acquired HIV-1 infection is critically influenced by age and the state of the CD4+ T cell compartment

The immunology of vertical HIV transmission differs from that of adult infection in that the immune system of the infant is not fully matured, and the factors that influence the functionality of CD8(+) T cell responses against HIV in children remain largely undefined. We have investigated CD8(+) T cell responses in 65 pediatric subjects with vertically acquired HIV-1 infection. Vigorous, broad, and Ag dose-driven CD8(+) T cell responses against HIV Ags were frequently observed in children who were older than 3 years of age and maintained CD4(+) T cell counts >400 cells/ micro l. In contrast, younger age or a CD4(+) T cell count <400 cells/ micro l was associated with poor CD8(+) T cell responses and high HIV loads. Furthermore, subjects with a severely depleted and phenotypically altered CD4(+) T cell compartment had circulating Gag-specific CD8(+) T cells with impaired IFN-gamma production. When viral load was not suppressed by antiviral treatment, subjects that fell below the putative age and CD4(+) T cell count thresholds had significantly reduced CD8(+) T cell responses and significantly higher viral loads. Thus, the data suggest that fully effective HIV-specific CD8(+) T cell responses take years to develop despite an abundance of Ag in early life, and responses are further severely impaired, independent of age, in children who have a depleted or skewed CD4(+) T cell compartment. The results are discussed in relation to differences between the neonatal and adult immune systems in the ability to respond to HIV infection.     

High Transmitter CD4+ T-Cell Count Shortly after the Time of Transmission in a Study of African Serodiscordant Couples

Background

2013 WHO guidelines recommend starting ART at CD4+ T-cell counts ≤500 cells/μL. We present the T-cell counts from adult Africans with HIV shortly following transmission to their sexual partners.

Methods

HIV-discordant couples in Zambia, Uganda and Rwanda were followed prospectively and received couples counseling and condoms. HIV uninfected partners were tested for HIV at least quarterly and HIV-infected partners received HIV care and referral for ART per national guidelines. Upon diagnosis of incident HIV infection in the previously HIV-uninfected partner, a blood sample was collected from both partners to measure CD4+ T-cells and perform viral linkage. The estimated date of infection (EDI) of the incident case was calculated based on testing history. EDI was unknown for suspected transmitting partners.

Results

From 2006–2011, 4,705 HIV-discordant couples were enrolled in this cohort, and 443 cases of incident HIV infection were documented. Virus linkage analysis was performed in 374 transmission pairs, and 273 (73%) transmissions were linked genetically. CD4 counts in the transmitting partner were measured a median of 56 days after EDI (mean:90.5, min:10, max:396). The median CD4 count was 339 cells/μl (mean:386.4, min:15, max:1,434), and the proportion of partners with a CD4+ T-cell count above 500/μl was 25% (95% CI:21, 31).

Conclusions

In our cohort of discordant couples, 73% of HIV transmissions occurred within the relationship, and the transmitter CD4+ T cell count shortly after the transmission event was frequently higher than the WHO 2013 ART-initiation guidelines.     

Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications

Clostridia are anaerobic Firmicutes producing a large array of metabolites by utilizing simple and complex carbohydrates, such as cellulose, as well as CO2/H2 or CO. Their exceptional substrate diversity is enhanced by their ability to produce a broad spectrum of chemicals that can be used as precursors to or directly as biofuels and industrial chemicals. Genetic and genomic tools are under intense development, and recent efforts to metabolically engineer clostridia demonstrate their potential for biofuel and biorefinery applications. Pathway engineering to combine established substrate-utilization programs, such as for cellulose, CO2/H2 or CO, with desirable metabolic programs could lead to modular design of strains suitable for many applications. Engineering complex phenotypes--aerotolerance, abolished sporulation, and tolerance to toxic chemicals--could lead to superior bioprocessing strains.     

A continuous, fluorescent, high-throughput assay for human dimethylarginine dimethylaminohydrolase-1

Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possess adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4446 compounds, and two representative hits are described, including a known DDAH-1 inhibitor. This is the most sensitive DDAH-1 assay reported to date and enables screening of compound libraries using [S] = K (M) conditions while displaying Z' factors from 0.6 to 0.8. Therefore, this strategy now makes possible high-throughput screening for human DDAH-1 inhibitors in pursuit of molecular probes and drugs to control excessive nitric oxide production.     

Overexpression of fetA (ybbL) and fetB (ybbM), Encoding an Iron Exporter,Enhances Resistance to Oxidative Stress in Escherichia coli

Reactive oxygen species are generated by redox reactions and the Fenton reaction of H₂O₂ and iron that generates the hydroxyl radical that causes severe DNA, protein, and lipid damage. We screened Escherichia coli genomic libraries to identify a fragment, containing cueR, ybbJ, qmcA, ybbL, and ybbM, which enhanced resistance to H₂O₂ stress. We report that the ΔybbL and ΔybbM strains are more susceptible to H₂O₂ stress than the parent strain and that ybbL and ybbM overexpression overcomes H₂O₂ sensitivity. The ybbL and ybbM genes are predicted to code for an ATP-binding cassette metal transporter, and we demonstrate that YbbM is a membrane protein. We investigated various metals to identify iron as the likely substrate of this transporter. We propose the gene names fetA and fetB (for Fe transport) and the gene product names FetA and FetB. FetAB allows for increased resistance to oxidative stress in the presence of iron, revealing a role in iron homeostasis. We show that iron overload coupled with H₂O₂ stress is abrogated by fetA and fetB overexpression in the parent strain and in the Δfur strain, where iron uptake is deregulated. Furthermore, we utilized whole-cell electron paramagnetic resonance to show that intracellular iron levels in the Δfur strain are decreased by 37% by fetA and fetB overexpression. Combined, these findings show that fetA and fetB encode an iron exporter that has a role in enhancing resistance to H₂O₂-mediated oxidative stress and can minimize oxidative stress under conditions of iron overload and suggest that FetAB facilitates iron homeostasis to decrease oxidative stress.     

Mechanistic similarity and diversity among the guanidine-modifying members of the pentein superfamily

The pentein superfamily is a mechanistically diverse superfamily encompassing both noncatalytic proteins and enzymes that catalyze hydrolase, dihydrolase and amidinotransfer reactions on guanidine substrates. Despite generally low sequence identity, they possess a conserved structural fold and display common mechanistic themes in catalysis. The structurally characterized catalytic penteins possess a conserved core of residues that include a Cys, His and two polar, guanidine-binding residues. All known catalytic penteins use the core Cys to attack the substrate's guanidine moiety to form a covalent thiouronium adduct and all cleave one or more of the guanidine C―N bonds. The mechanistic information compiled to date supports the hypothesis that this superfamily may have evolved divergently from a catalytically promiscuous ancestor.