Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.
Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of ε-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher kcat, but Km also increased so the specificity constant, kcat/Km, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of ε-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds ε-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.
(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate (HG). This reaction involves decarboxylation, substituent migration, and aromatic oxygenation in a single catalytic cycle. HPPD is a unique member of the α-keto acid dependent oxygenases that require Fe(II) and an α-keto acid substrate to oxygenate or oxidize an organic molecule. We have examined the reaction coordinate of HPPD from Streptomyces avermitilis using rapid mixing pre-steady-state methods in conjunction with steady-state kinetic analyses. Acid quench reactions and product analysis of homogentisate indicate that HPPD as isolated is fully active and that experiments limited in dioxygen concentration with respect to that of the enzyme do involve a single turnover. These experiments indicate that during the course of one turnover the concentration of homogentisate is stoichiometric with enzyme concentration by ∼200 ms, well before the completion of the catalytic cycle. Subsequent single turnover reactions were monitored spectrophotometrically under pseudo-first-order and matched concentration reactant conditions. Three spectrophotometrically distinct intermediates are observed to accumulate. The first of these is a relatively strongly absorbing species with maxima at 380 and 480 nm that forms with a rate constant (k1) of 7.4 × 104 M-1 s-1 and then decays to a second intermediate with a rate constant (k2) of 74 s-1. The rate constant for the decay of the second intermediate (k3) is 13 s-1 and is concomitant with the formation of the product, homogentisate, based on rapid quench and pre-steady-state fluorescence measurements. The rate constant for this process decreases to 7.6 s-1 when deuterons are substituted for protons in the aromatic ring of the substrate. The release of product from the enzyme is rate limiting and occurs at 1.6 s-1. This final event exhibits a kinetic isotope effect of 2 with deuterium oxide as the solvent, consistent with a solvent isotope effect on Vmax of 2.6 observed in steady-state experiments.
(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) incorporates both atoms of molecular oxygen into 4-hydroxyphenylpyruvate (HPP) to form homogentisate (HG). This reaction has direct relevance in both medicine and agriculture. In humans, the specific inhibition of HPPD alleviates the symptoms of diseases that arise from tyrosine catabolism defects. However, in plants, the inhibition of HPPD bleaches, stunts, and ultimately kills the organism. The reason for this is that in mammalian metabolism the product HG does not feed into other pathways, whereas in plants it is the precursor for the redox active portion of tocopherols and plastoquinones. There are a number of commercially available herbicides that directly target the inhibition of the HPPD reaction. Plant HPPD however is largely uncharacterized in terms of its catalysis and inhibition reactions. In this study, we examine the catalysis and inhibition of HPPD from Arabidopsis thaliana (AtHPPD). We have expressed AtHPPD and purified the enzyme to high specific activity. This form of HPPD accumulates two transient species in single turnover reactions with the native substrate HPP. These transients appear to be equivalent to intermediates I and III observed in the enzyme from Streptomyces (Johnson-Winters et al. (2005), Biochemistry, 44, 7189−7199). The first intermediate is a relatively strongly absorbing species with maxima at 380 and 490 nm. This species decays to a second intermediate that is fluorescent and has been assigned as the complex of the enzyme with the product, HG. The decay of this intermediate is rate-determining in multiple turnover reactions. The reaction of the enzyme with the analogue of the substrate, phenylpyruvate (PPA), is noncatalytic. A single turnover reaction is observed with this ligand that renders the enzyme oxidized to the ferric form, consumes a stoichiometric amount of dioxygen, and yields 66% phenylacetate as a product. Additional absorbance features at 365 and 670 nm accumulate during inactivation and give the inactivated enzyme a green color but has the same molecular mass as the active enzyme as determined by mass spectrometry.
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