Zhuang Z.,University of New Mexico |
Zhuang Z.,University of Delaware |
Latham J.,University of New Mexico |
Song F.,University of New Mexico |
And 5 more authors.
Biochemistry | Year: 2012
The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [ 14C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K is = 250 ± 70 μM; K ii = 900 ± 300 μM) and 4-HB (K is = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D 2O solvent kinetic isotope effect (3.8) on the steady-state k cat value (18 s -1) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [ 14C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [ 14C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent 18O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4- hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom. © 2011 American Chemical Society.
Deshpande A.,Los Alamos National Laboratory |
Gans J.,Los Alamos National Laboratory |
Graves S.W.,University of New Mexico |
Green L.,Los Alamos National Laboratory |
And 9 more authors.
Journal of Microbiological Methods | Year: 2010
We have developed a rapid (under 4 hours), multiplex, nucleic acid assay, adapted to a microsphere array detection platform. We call this assay multiplex oligonucleotide ligation-PCR (MOL-PCR). Unlike other ligation-based assays that require multiple steps, our protocol consists of a single tube reaction, followed by hybridization to a Luminex microsphere array for detection. We demonstrate the ability of this assay to simultaneously detect diverse nucleic acid signatures (e.g., unique sequences, single nucleotide polymorphisms) in a single multiplex reaction. Detection probes consist of modular components that enable target detection, probe amplification, and subsequent capture onto microsphere arrays. To demonstrate the utility of our assay, we applied it to the detection of three biothreat agents, B. anthracis, Y. pestis, and F. tularensis. Combined with the ease and robustness of this assay, the results presented here show a strong potential of our assay for use in diagnostics and surveillance.
Song F.,University of New Mexico |
Thoden J.B.,University of Wisconsin - Madison |
Zhuang Z.,University of New Mexico |
Latham J.,University of New Mexico |
And 3 more authors.
Biochemistry | Year: 2012
The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to 18O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from 31P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring. © 2012 American Chemical Society.
Hwang R.-J.,Scientific Laboratory Division |
Beltran J.,Scientific Laboratory Division |
Rogers C.,Scientific Laboratory Division |
Barlow J.,Scientific Laboratory Division |
Razatos G.,Scientific Laboratory Division
Journal of Forensic Sciences | Year: 2016
Aqueous ethanol wet-bath simulator solutions are used to perform calibration adjustments, calibration checks, proficiency testing, and inspection of breath alcohol instruments. The Toxicology Bureau of the New Mexico Department of Health has conducted a study to estimate a measurement of uncertainty for the preparation and testing of these wet-bath simulator solutions. The measurand is identified as the mass concentration of ethanol (g/100 mL) determined through dual capillary column headspace gas chromatography with flame ionization detector analysis. Three groups were used in the estimation of the aqueous ethanol wet-bath simulator solutions uncertainty: GC calibration adjustment, GC analytical, and certified reference material. The standard uncertainties for these uncertainty sources were combined using the method of root-sum-squares to give uc = 0.8598%. The combined standard uncertainty was expanded to U = 1.7% to reflect a confidence level of 95% using a coverage factor of 2. This estimation applies to all aqueous ethanol wet-bath simulator solution concentrations produced by this laboratory. © 2016 American Academy of Forensic Sciences
Pesko K.,Yale University |
Pesko K.,Scientific Laboratory Division |
Voigt E.A.,University of Wisconsin - Madison |
Swick A.,University of Wisconsin - Madison |
And 5 more authors.
Frontiers in Genetics | Year: 2015
Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5' end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene order. © 2015 Pesko, Voigt, Swick, Morley, Timm, Yin and Turner.
Krinsky C.S.,University of New Mexico |
Lathrop S.L.,University of New Mexico |
Crossey M.,TriCore Reference Laboratories |
Baker G.,Scientific Laboratory Division |
Zumwalt R.,University of New Mexico
American Journal of Forensic Medicine and Pathology | Year: 2011
Since its approval in the United States, fentanyl has become increasingly popular for the medical management of pain and as a substance of abuse. Fentanyl is unique among the opioids in its widespread use with a transdermal delivery system, which contributes to its unique pharmacokinetics and abuse potential. We examined the demographics of deaths with fentanyl identified on toxicologic analysis and reviewed specific challenges in the laboratory detection of postmortem fentanyl levels. The New Mexico Office of the Medical Investigator database was searched for all cases from January 1986 through December 2007 with fentanyl reported as present or quantified. Those deaths with a cause of death identified as drug overdose were then analyzed separately. From 1986 to 2007, 154 cases were identified with fentanyl present in postmortem samples, with 96 of the cases identified as fentanyl-related drug overdoses. The number of fentanyl-related deaths has increased over the past 20 years, corresponding to both statewide increases in the medical use of fentanyl and the abuse of prescription opioids. The demographics of these fentanyl-related overdoses showed that subjects were more likely to be female, white non-Hispanic, and older than those in previously described overdose deaths. Several cases were identified with central and peripheral blood samples and antemortem and postmortem samples available for fentanyl quantification. Given the uncharacteristic demographics of fentanyl-related deaths and the complexity of the laboratory analysis of fentanyl, forensic scientists must use caution in both the detection and interpretation of fentanyl concentrations. Copyright © 2011 by Lippincott Williams & Wilkins.
Khan M.M.T.,University of New Mexico |
Chapman T.,Scientific Laboratory Division |
Cochran K.,Scientific Laboratory Division |
Schuler A.J.,University of New Mexico
Water Research | Year: 2013
Submerged biofilm systems, such as integrated fixed-film activated sludge (IFAS) and moving bed bioreactors (MBBRs), are increasingly being used for domestic wastewater treatment, often to improve nitrification. Little is known about whether and how biofilm attachment surface chemical properties affect treatment performance, although surface chemistry is known to affect attachment in other systems, and work with pure strains has suggested that attachment of nitrifying bacteria may be enhanced on high surface energy surfaces. The objective of this research was to systematically evaluate the effects of surface chemistry on biofilm quantity and rates of nitrification and estrogen removal. Biofilms were grown on four plastic attachment surfaces with a range of hydrophobicity and surface energy values (nylon, melamine, high-density-polyethylene [HDPE], and acetal polymeric plastic) by immersing them in a full scale nitrifying activated sludge wastewater treatment system, followed by batch test experiments. The attachment surface water contact angles ranged from 53° to 98° and surface energies ranged from 48.9 to 20.9 mJ/m2. Attachment surface hydrophilicity and surface energy were positively correlated with total biomass attachment, with more than twice as much biomass on the highest surface energy, most hydrophilic surface (nylon) than on the lowest surface energy, least hydrophilic surface (acetal plastic). Absolute and specific nitrification rates were also correlated with hydrophilicity and surface energy (varying by factors of 5 and 2, respectively), as were absolute and specific removal first order rate constants of the hormones estrone (E1), β-estradiol (E2) and 17α-ethynylestradiol (EE2). These results suggested that attachment surface chemistry may be a useful design parameter for improving biofilm performance for removal of ammonia and endocrine disrupting hormones from wastewater. Further research is required to verify these results at longer time scales and with typical media geometries. © 2013 Elsevier Ltd.
PubMed | Scientific Laboratory Division
Type: Journal Article | Journal: Journal of forensic sciences | Year: 2016
Aqueous ethanol wet-bath simulator solutions are used to perform calibration adjustments, calibration checks, proficiency testing, and inspection of breath alcohol instruments. The Toxicology Bureau of the New Mexico Department of Health has conducted a study to estimate a measurement of uncertainty for the preparation and testing of these wet-bath simulator solutions. The measurand is identified as the mass concentration of ethanol (g/100 mL) determined through dual capillary column headspace gas chromatography with flame ionization detector analysis. Three groups were used in the estimation of the aqueous ethanol wet-bath simulator solutions uncertainty: GC calibration adjustment, GC analytical, and certified reference material. The standard uncertainties for these uncertainty sources were combined using the method of root-sum-squares to give uc = 0.8598%. The combined standard uncertainty was expanded to U = 1.7% to reflect a confidence level of 95% using a coverage factor of 2. This estimation applies to all aqueous ethanol wet-bath simulator solution concentrations produced by this laboratory.