Search

Current lye processing for debittering California black table olives produces large amounts of caustic wastewater and destroys many of the beneficial phenolic compounds in the fruit. Herein, we propose using enzyme treatment in place of lye, potentially reducing the amount and causticity of wastewater produced. By specifically targeting the bitterness-causing compound, oleuropein, retention of other beneficial phenolics may be possible. A β-glucosidase from Streptomyces sp. was identified from a screen of 22 glycosyl hydrolases to completely degrade oleuropein in 24 h. Computational modeling was performed on this enzyme, and mutation C181A was found to improve the rate of catalysis by 3.2-fold. This mutant was tested in the context of the olive fruit and leaf extract. Degradation was observed in the olive leaf extract but not in the fruit matrix, suggesting that enzyme fruit penetration is a limiting factor. This work discovers and begins the refinement process for an enzyme that has the catalytic properties for debittering olives and provides direction for future engineering efforts required to make a product with commercial value.

A 3D molecular arrangement and coordination of a minor anthocyanin from red cabbage creates a brilliant blue color., The color of food is critical to the food and beverage industries, as it influences many properties beyond eye-pleasing visuals including flavor, safety, and nutritional value. Blue is one of the rarest colors in nature’s food palette—especially a cyan blue—giving scientists few sources for natural blue food colorants. Finding a natural cyan blue dye equivalent to FD&C Blue No. 1 remains an industry-wide challenge and the subject of several research programs worldwide. Computational simulations and large-array spectroscopic techniques were used to determine the 3D chemical structure, color expression, and stability of this previously uncharacterized cyan blue anthocyanin-based colorant. Synthetic biology and computational protein design tools were leveraged to develop an enzymatic transformation of red cabbage anthocyanins into the desired anthocyanin. More broadly, this research demonstrates the power of a multidisciplinary strategy to solve a long-standing challenge in the food industry.

The identification of environmental factors that lead to loss of tolerance has been coined the holy grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein, we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177-314 of PDC-E2 by joining a recombinant peptide spanning residues 177-252 (PDC-228) with a 62-residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 inner lipoyl domain (ILD). We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA-modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the immunoglobulin M isotype and more dominant in early-stage primary biliary cholangitis (PBC), suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition, we analyzed PPL, LA-PPL, and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by enzyme-linked immunosorbent assay, immunoblotting, and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid. Conclusion: A molecular understanding of the conformation of xenobiotic-modified PDC-E2 is critical for understanding xenobiotic modification and loss of tolerance in PBC with widespread implications for a role of environmental chemicals in the induction of autoimmunity. (Hepatology 2017;65:1670-1682).

Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8-bis(acetylthio) octanoic acid (SAc)-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme-linked immunosorbent assay (ELISA); SAc conjugate and rPDC-E2-specific affinity-purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late-stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC-E2 in early-stage versus late-stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. Conclusion: Specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e., by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host. (HEPATOLOGY 2013)

Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces anti-mitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl moiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC.

An operationally simple, one-pot, two-step cascade method has been developed to afford quinazolino[1,2,3]triazolo[1,4]benzodiazepines. This unique, atom-economical transformation engages five reactive centers (amide, aniline, carbonyl, azide, and alkyne) and employs environmentally benign iodine as a catalyst. The method proceeds via sequential quinazolinone-forming condensation and intramolecular azide–alkyne 1,3-dipolar cycloaddition reactions. Substrate scope, multicomponent examples, and mechanistic insights are discussed.

An operationally simple, one-pot, two-step cascade method has been developed to afford quinazolino[1,2,3]triazolo[1,4]benzodiazepines. This unique, atom-economical transformation engages five reactive centers (amide, aniline, carbonyl, azide, and alkyne) and employs environmentally benign iodine as a catalyst. The method proceeds via sequential quinazolinone-forming condensation and intramolecular azide–alkyne 1,3-dipolar cycloaddition reactions. Substrate scope, multicomponent examples, and mechanistic insights are discussed.

Routes to structurally unique spiro-fused pyrazolidoylisoxazolines are reported. These methods start with monosubstituted hydrazines or hydrazides and utilize the nitrile oxide 1,3-dipolar cycloaddition reaction to generate the targeted spiro-fused bis-heterocycles. Molecular shape space diversity analyses were performed on these pyrazolidoylisoxazolines showing that manipulation of the appended R groups significantly changes the molecular shape.

Chorismate-utilizing enzymes are attractive antimicrobial drug targets due to their absence in humans and their central role in bacterial survival and virulence. The structural and mechanistic homology of a group of these inspired the goal of discovering inhibitors that target multiple enzymes. Previously, we discovered seven inhibitors of 4-amino-4-deoxychorismate synthase (ADCS) in an on-bead, fluorescent-based screen of a 2304-member one-bead-one-compound combinatorial library. The inhibitors comprise PAYLOAD and COMBI stages, which interact with active site and surface residues, respectively, and are linked by a SPACER stage. These seven compounds, and six derivatives thereof, also inhibit two other enzymes in this family, isochorismate synthase (IS) and anthranilate synthase (AS). The best binding compound inhibits ADCS, IS, and AS with K(i) values of 720, 56, and 80 microM, respectively. Inhibitors with varying SPACER lengths show the original choice of lysine to be optimal. Lastly, inhibition data confirm the PAYLOAD stage directs the inhibitors to the ADCS active site.