Purification, Characterization, and Structural Studies of a Sulfatase from .

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications.

Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives.

Next generation β-glucuronidases can effectively cleave glucuronides in urine at room temperature. However, during the discovery studies, additional challenges were identified for urine drug testing across biologically relevant pH extremes and patient urine specimens. Different enzymes were evaluated across clinical urine specimens and commercially available urine control matrices.

Variants of glycosyl hydrolase family 2 β-glucuronidases have increased activity on recalcitrant substrates.

Glucuronidated drug metabolites can be quantified from urine samples by first hydrolyzing conjugates with β-glucuronidase (β-GUS) and then separating free drug molecules by liquid chromatography and mass spectrometry detection (LC-MS). To improve the activity and specificity of various β-GUS, we designed enzyme chimeras and generated site-saturation variants based on structural analyses, then screened them for improved activity on drug metabolites important to clinical and forensic drug-testing laboratories. Often, an increase of activity on one substrate of interest was countered by loss of activity against another, and there was no strong correlation of activity on standard β-glucuronidase substrates to activity on recalcitrant drug glucuronides.

Parallel sample processing using dispersive INtip micro-purification on programmable multichannel pipettes.

Automation gives researchers the ability to process and screen orders of magnitude higher numbers of samples than manual experimentation. Current biomacromolecule separation methodologies suffer from necessary manual intervention, making their translation to high-throughput automation difficult. Herein, we present the first characterization of biomacromolecule affinity purification via dispersive solid-phase extraction in a pipette tip (INtip).

Survey of Candidate Genes for Maize Resistance to Infection by Aspergillus flavus and/or Aflatoxin Contamination.

Many projects have identified candidate genes for resistance to aflatoxin accumulation or infection and growth in maize using genetic mapping, genomics, transcriptomics and/or proteomics studies. However, only a small percentage of these candidates have been validated in field conditions, and their relative contribution to resistance, if any, is unknown. This study presents a consolidated list of candidate genes identified in past studies or in-house studies, with descriptive data including genetic location, gene annotation, known protein identifiers, and associated pathway information, if known.

Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities.

Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon ((12)C) or stable-isotope-labeled ((13)C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose).

Stoichiometry of energy coupling by proton-translocating ATPases: a history of variability.

One of the central energy-coupling reactions in living systems is the intraconversion of ATP with a transmembrane proton gradient, carried out by proton-translocating F- and V-type ATPases/synthases. These reversible enzymes can hydrolyze ATP and pump protons, or can use the energy of a transmembrane proton gradient to synthesize ATP from ADP and inorganic phosphate. The stoichiometry of these processes (H(+)/ATP, or coupling ratio) has been studied in many systems for many years, with no universally agreed upon solution.

The Escherichia coli F1F0 ATP synthase displays biphasic synthesis kinetics.

The F1F0 proton-translocating ATPase/synthase is the primary generator of ATP in most organisms growing aerobically. Kinetic assays of ATP synthesis have been conducted using enzymes from mitochondria and chloroplasts. However, limited data on ATP synthesis by the model Escherichia coli enzyme are available, mostly because of the lack of an efficient and reproducible assay.

Characterization of reconstituted Fo from wild-type Escherichia coli and identification of two other fluxes co-purifying with Fo.

We purified the ATPase Fo sector from a nonoverexpressing strain of Escherichia coli, reconstituted it into lipid vesicles made of either asolectin or two different mixtures of purified lipids, and measured proton flux through the reconstituted proton channel. We measured single-channel conductances and found that Fo activity depends on both lipids and reconstitution methods. In asolectin vesicles, Fo has a single-channel conductance of about 0.

A functional His-tagged c subunit of the Escherichia coli F-type ATPase/Synthase.

The c subunit of the Escherichia coli F0 has been tagged with a hexahistidine motif at its C-terminus. The tagged subunit is capable of forming functional F0 complexes that translocate protons in the absence of the F1 complex. In the presence of F1, the two sectors associate and display all biochemical activities of the wildtype enzyme: DCCD-inhibitable ATPase activity, ATP synthase activity, and ATP-dependent proton pumping.

Purification and ligand binding of EmrR, a regulator of a multidrug transporter.

EmrR, the repressor of the emrRAB operon of Escherichia coli, was purified to 95% homogeneity. EmrR was found to bind putative ligands of the EmrAB pump-2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone-with affinities in the micromolar range. Equilibrium dialysis experiments suggested one bound ligand per monomer of the dimeric EmrR.

V1-situated stalk subunits of the yeast vacuolar proton-translocating ATPase.

The proton-translocating ATPase of the yeast vacuole is an enzyme complex consisting of a large peripheral membrane sector (V1) and an integral membrane sector (V0), each composed of multiple subunits. The V1 sector contains subunits that hydrolyze ATP, whereas the V0 sector contains subunits that translocate protons across the membrane. Additional subunits in both sectors couple these activities.

Reconstitution in vitro of the V1 complex from the yeast vacuolar proton-translocating ATPase. Assembly recapitulates mechanism.

Oligomeric assembly is a fundamental aspect of many complex enzymes. Using our native gel technique for examining subcomplexes of the V-ATPase V1 sector, we have developed an in vitro reconstitution assay for assembly of this complex. Assembly of complex II, the soluble V1 complex observed in native gels, is dependent upon the presence of divalent cations and physiological temperatures.

Resolution of subunit interactions and cytoplasmic subcomplexes of the yeast vacuolar proton-translocating ATPase.

The vacuolar proton-translocating ATPase is the principal energization mechanism that enables the yeast vacuole to perform most of its physiological functions. We have undertaken an examination of subunit-subunit interactions and assembly states of this enzyme. Yeast two-hybrid data indicate that Vma1p and Vma2p interact with each other and that Vma4p interacts with itself.

Analysis of estrogen receptor interaction with tertiary-structured estrogen responsive elements.

An initial crucial step in estrogen activation of gene expression is the interaction of the estrogen receptor with a specific nucleotide sequence [estrogen responsive element (ERE)]. Previously, we found that the estrogen receptor binds preferentially and with high affinity to the lower strand of the rat prolactin imperfect ERE which contains tertiary structure (Lannigan DA and Notides AC, Proc Natl Acad Sci USA 86: 863-867, 1989). Using perfect and imperfect EREs from the upstream region of the chicken vitellogenin II gene, we have now extended our findings and have determined that the estrogen receptor preferentially interacts with either perfect or imperfect EREs which contain tertiary structure.