Novel Findings regarding the Bioactivity of the Natural Blue Pigment Genipin in Human Diseases

Genipin is an important monoterpene iridoid compound isolated from Gardenia jasminoides J.Ellis fruits and from Genipa americana fruits, or genipap. It is a precursor of a blue pigment which may be attractive alternative to existing food dyes and it possesses various potential therapeutic properties such as anti-cancer, anti-diabetic and hepatoprotective activity. Biomedical studies also show that genipin may act as a neuroprotective drug. This review describes new aspects of the bioactivity of genipin against various diseases, as well as its toxicity and industrial applications, and presents its potential mechanism of action.

Reprogramming Versus Subdomain Substitution: An Exploration of Strategies for NRPS Engineering Using the Unique Model System BpsA

<p>Non-ribosomal peptide synthetases (NRPSs) are large enzymes that generate a plethora of important natural products, from antibiotics to immunosuppressants. These modular enzymes function like an assembly line, selecting and incorporating specific (and frequently nonproteinogenic) amino acids into a growing peptide chain. This modular structure offers promise for re-engineering NRPS units to generate new useful products, but progress has to date been limited by the complex and dynamic nature of key domains, and a failure to define generally applicable “rules” to guide engineering efforts. Early efforts to engineer NRPS enzymes relied on the substitution of entire NRPS modules or domains, but product yields were often very low. However, these studies did highlight the promise of targeting the adenylation domain, the part of each NRPS modules that is responsible for selecting each amino acid substrate. Two particularly promising strategies for NRPS engineering aim to manipulate the adenylation domain in ways that minimise steric disruption to the assembly line. The first of these, reprogramming, makes the fewest possible changes to the NRPS primary sequence, but is dependent on those precise changes conforming to the existing structure of the adenylation domain binding pocket. More recently a second technique has been developed, subdomain substitution, which recombines a larger region of the adenylation domain to avoid perturbation of the binding pocket. The research described in this thesis examined and compared both approaches using the unique NRPS BpsA as a model system. BpsA is a single-module NRPS that generates a vivid blue pigment product, making for a reductionist system that offers a robust visual reporter capacity. Experiments with the reprogramming technique showed that small changes to the protein sequence had potential to exert major impacts on enzyme function, even when no change to function was intended. In contrast, experiments with subdomain substitution were generally more effective, showing that NRPS enzymes are very sensitive to the precise boundaries of the substituted region, but that activity can be restored to otherwise non-functional subdomain substitutions by modulation of the regional boundaries.</p>

Reprogramming Versus Subdomain Substitution: An Exploration of Strategies for NRPS Engineering Using the Unique Model System BpsA

<p>Non-ribosomal peptide synthetases (NRPSs) are large enzymes that generate a plethora of important natural products, from antibiotics to immunosuppressants. These modular enzymes function like an assembly line, selecting and incorporating specific (and frequently nonproteinogenic) amino acids into a growing peptide chain. This modular structure offers promise for re-engineering NRPS units to generate new useful products, but progress has to date been limited by the complex and dynamic nature of key domains, and a failure to define generally applicable “rules” to guide engineering efforts. Early efforts to engineer NRPS enzymes relied on the substitution of entire NRPS modules or domains, but product yields were often very low. However, these studies did highlight the promise of targeting the adenylation domain, the part of each NRPS modules that is responsible for selecting each amino acid substrate. Two particularly promising strategies for NRPS engineering aim to manipulate the adenylation domain in ways that minimise steric disruption to the assembly line. The first of these, reprogramming, makes the fewest possible changes to the NRPS primary sequence, but is dependent on those precise changes conforming to the existing structure of the adenylation domain binding pocket. More recently a second technique has been developed, subdomain substitution, which recombines a larger region of the adenylation domain to avoid perturbation of the binding pocket. The research described in this thesis examined and compared both approaches using the unique NRPS BpsA as a model system. BpsA is a single-module NRPS that generates a vivid blue pigment product, making for a reductionist system that offers a robust visual reporter capacity. Experiments with the reprogramming technique showed that small changes to the protein sequence had potential to exert major impacts on enzyme function, even when no change to function was intended. In contrast, experiments with subdomain substitution were generally more effective, showing that NRPS enzymes are very sensitive to the precise boundaries of the substituted region, but that activity can be restored to otherwise non-functional subdomain substitutions by modulation of the regional boundaries.</p>

Engineering the indigoidine-synthesising enzyme BpsA for diverse applications in biotechnology

<p>Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that synthesise bioactive peptides using an assembly line architecture, wherein each module is responsible for the incorporation of a monomer into the growing chain. Present in both fungi and bacteria, NRPSs are responsible for a wide variety of secondary metabolites and bioactive compounds including siderophores, antibiotics, anti-cancer compounds and immunosuppressants. For functionality, NRPSs require the attachment of a phosphopantetheine moiety to their peptidyl carrier protein domains. This reaction is catalysed by a phosphopantetheinyl transferase (PPTase). The NRPS blue pigment synthetase A (BpsA) is unusual in that it is comprised of only a single module. BpsA contains an adenylation domain that recognises and sequentially binds two molecules of L-glutamine, an oxidation domain that is believed to oxidise each glutamine monomer, a peptidyl carrier protein domain that binds the phosphopantetheine moiety, and a thioesterase domain that cyclises each glutamine and releases the final bicyclic product from the enzyme. This final product is a blue pigment called indigoidine, and its synthesis from two molecules of L-glutamine is powered by ATP. Comparatively to other NRPSs BpsA is easy to manipulate and work with both in vitro and in vivo. Here, the ability to easily detect synthesis of indigoidine was utilised to provide a versatile reporter to detect a variety of biochemical activities. PPTases are essential enzymes that are promising drug targets in the clinically important bacteria Pseudomonas aeruginosa and Mycobacterium tuberculosis. BpsA can be purified in the inactive apo form, which then requires a PPTase to activate it to enable indigoidine synthesis. Here it was shown that mixing BpsA, a PPTase, the enzymatic substrates, and a potential inhibitor enables screening for PPTase inhibition by monitoring the rate or extent of indigoidine synthesis. This method was optimised and used to screen commercial drug libraries against two PPTases, PcpS from P. aeruginosa and PptT from M. tuberculosis. Several novel inhibitors were identified and pilot in vivo studies were performed. M. tuberculosis also possesses a second essential PPTase called TB-AcpS, which has very narrow substrate specificity and cannot post-translationally modify BpsA. In an attempt to widen the substrate specificity a combination of rational engineering and directed evolution was employed. These attempts did not yield significant improvements in the ability of TB-AcpS to activate modified BpsA, however they did yield mutants that were more effective substrates for other type I PPTases. The easily detectable nature of indigoidine also enabled application of BpsA as a reporter for a range of different substrates. Particularly effective was development of a commercially applicable method using BpsA to quantify L-glutamine in a range of conditions, including cell culture media and blood. The assay developed offers several advantages over currently available kits. BpsA was also used to detect and quantify ATP, and this was applied to monitor adenylation reactions. Finally, the ability of BpsA to synthesise indigoidine-like compounds from glutamine analogues was explored.</p>

Engineering the indigoidine-synthesising enzyme BpsA for diverse applications in biotechnology

<p>Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that synthesise bioactive peptides using an assembly line architecture, wherein each module is responsible for the incorporation of a monomer into the growing chain. Present in both fungi and bacteria, NRPSs are responsible for a wide variety of secondary metabolites and bioactive compounds including siderophores, antibiotics, anti-cancer compounds and immunosuppressants. For functionality, NRPSs require the attachment of a phosphopantetheine moiety to their peptidyl carrier protein domains. This reaction is catalysed by a phosphopantetheinyl transferase (PPTase). The NRPS blue pigment synthetase A (BpsA) is unusual in that it is comprised of only a single module. BpsA contains an adenylation domain that recognises and sequentially binds two molecules of L-glutamine, an oxidation domain that is believed to oxidise each glutamine monomer, a peptidyl carrier protein domain that binds the phosphopantetheine moiety, and a thioesterase domain that cyclises each glutamine and releases the final bicyclic product from the enzyme. This final product is a blue pigment called indigoidine, and its synthesis from two molecules of L-glutamine is powered by ATP. Comparatively to other NRPSs BpsA is easy to manipulate and work with both in vitro and in vivo. Here, the ability to easily detect synthesis of indigoidine was utilised to provide a versatile reporter to detect a variety of biochemical activities. PPTases are essential enzymes that are promising drug targets in the clinically important bacteria Pseudomonas aeruginosa and Mycobacterium tuberculosis. BpsA can be purified in the inactive apo form, which then requires a PPTase to activate it to enable indigoidine synthesis. Here it was shown that mixing BpsA, a PPTase, the enzymatic substrates, and a potential inhibitor enables screening for PPTase inhibition by monitoring the rate or extent of indigoidine synthesis. This method was optimised and used to screen commercial drug libraries against two PPTases, PcpS from P. aeruginosa and PptT from M. tuberculosis. Several novel inhibitors were identified and pilot in vivo studies were performed. M. tuberculosis also possesses a second essential PPTase called TB-AcpS, which has very narrow substrate specificity and cannot post-translationally modify BpsA. In an attempt to widen the substrate specificity a combination of rational engineering and directed evolution was employed. These attempts did not yield significant improvements in the ability of TB-AcpS to activate modified BpsA, however they did yield mutants that were more effective substrates for other type I PPTases. The easily detectable nature of indigoidine also enabled application of BpsA as a reporter for a range of different substrates. Particularly effective was development of a commercially applicable method using BpsA to quantify L-glutamine in a range of conditions, including cell culture media and blood. The assay developed offers several advantages over currently available kits. BpsA was also used to detect and quantify ATP, and this was applied to monitor adenylation reactions. Finally, the ability of BpsA to synthesise indigoidine-like compounds from glutamine analogues was explored.</p>

Preparation and Characterization of Water-Insoluble Gardenia Blue Pigment

Based on molecular simulations, the synthetic route of water-insoluble gardenia blue pigment was prepared by the reaction of genipin and L-Phenylalanine methyl ester hydrochloride. A highly purified pigment was obtained after extraction by chloroform and purification by silica gel column chromatography, and the value of color is up to 288. A study on the structural characteristics of the pigment was implemented with a scanning electron microscope, ultraviolet-visible spectrophotometer, Fourier transform infrared spectrometer, X-ray photoelectron spectrometer, and quatropde-time of flight mass spectrometer. The results showed that the surface of the pigment was largely smooth and spherical; The λmax was 607 nm, and the main functional groups include O-C=O, C=O, C-N, C=C, OH, and benzene ring; We detrained six different molecular weight and chemical structures of pigments and speculated the particular structures and formation mechanisms of three kinds of pigment, whose molecular weights are 690.1156, 720.1226, and 708.1246 Da, respectively. The pigment was only able to be dissolved in ethanol, methanol, acetone, ethyl acetate, and other strong polar organic solvents, but was not able to be dissolved in water, ethyl ether, petroleum ether, and other weak polar organic solvents. In terms of light and thermal stabilities, water-insoluble gardenia blue pigment is significantly better than water-soluble gardenia blue pigment (p < 0.05). When it is under direct light for 7 days or incubated at 80–120 °C for 24 h, the pigment residual rates were 74.90, 95.26, 88.27, and 87.72%, respectively.

Non-conventional octameric structure of C-phycocyanin

C-phycocyanin (CPC), a blue pigment protein, is an indispensable component of giant phycobilisomes, which are light-harvesting antenna complexes in cyanobacteria that transfer energy efficiently to photosystems I and II. X-ray crystallographic and electron microscopy (EM) analyses have revealed the structure of CPC to be a closed toroidal hexamer by assembling two trimers. In this study, the structural characterization of non-conventional octameric CPC is reported for the first time. Analyses of the crystal and cryogenic EM structures of the native CPC from filamentous thermophilic cyanobacterium Thermoleptolyngbya sp. O–77 unexpectedly illustrated the coexistence of conventional hexamer and novel octamer. In addition, an unusual dimeric state, observed via analytical ultracentrifugation, was postulated to be a key intermediate structure in the assemble of the previously unobserved octamer. These observations provide new insights into the assembly processes of CPCs and the mechanism of energy transfer in the light-harvesting complexes.