Sparse Epistatic Patterns in the Evolution of Terpene Synthases.

We explore sequence determinants of enzyme activity and specificity in a major enzyme family of terpene synthases. Most enzymes in this family catalyze reactions that produce cyclic terpenes-complex hydrocarbons widely used by plants and insects in diverse biological processes such as defense, communication, and symbiosis. To analyze the molecular mechanisms of emergence of terpene cyclization, we have carried out in-depth examination of mutational space around (E)-ß-farnesene synthase, an Artemisia annua enzyme which catalyzes production of a linear hydrocarbon chain. Each mutant enzyme in our synthetic libraries was characterized biochemically, and the resulting reaction rate data were used as input to the Michaelis-Menten model of enzyme kinetics, in which free energies were represented as sums of one-amino-acid contributions and two-amino-acid couplings. Our model predicts measured reaction rates with high accuracy and yields free energy landscapes characterized by relatively few coupling terms. As a result, the Michaelis-Menten free energy landscapes have simple, interpretable structure and exhibit little epistasis. We have also developed biophysical fitness models based on the assumption that highly fit enzymes have evolved to maximize the output of correct products, such as cyclic products or a specific product of interest, while minimizing the output of byproducts. This approach results in nonlinear fitness landscapes that are considerably more epistatic. Overall, our experimental and computational framework provides focused characterization of evolutionary emergence of novel enzymatic functions in the context of microevolutionary exploration of sequence space around naturally occurring enzymes.

Discovery of germacrene A synthases in Barnadesia spinosa: The first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae.

The Andes-endemic Barnadesioideae lineage is the oldest surviving and phylogenetically basal subfamily of the Asteraceae (Compositae), a prolific group of flowering plants with world-wide distribution (∼24,000 species) marked by a rich diversity of sesquiterpene lactones (STLs). Intriguingly, there is no evidence that members of the Barnadesioideae produce STLs, specialized metabolites thought to have contributed to the adaptive success of the Asteraceae family outside South America. The biosynthesis of STLs requires the intimate expression and functional integration of germacrene A synthase (GAS) and germacrene A oxidase (GAO) to sequentially cyclize and oxidize farnesyl diphosphate into the advanced intermediate germacrene A acid leading to diverse STLs. Our previous discovery of GAO activity conserved across all major subfamilies of Asteraceae, including the phylogenetically basal lineage of Barnadesioideae, prompted further investigation of the presence of the gateway GAS in Barnadesioideae. Herein we isolated two terpene synthases (BsGAS1/BsGAS2) from the basal Barnadesia spinosa (Barnadesioideae) that displayed robust GAS activity when reconstituted in yeast and characterized in vitro. Despite the apparent lack of STLs in the Barnadesioideae, this work unambiguously confirms the presence of GAS in the basal genera of the Asteraceae. Phylogenetic analysis reveals that the two BsGASs fall into two distinct clades of the Asteraceae's GASs, and BsGAS1 clade is only retained in the evolutionary closer Cichorioideae subfamily, implicating BsGAS2 is likely the ancestral base of most GASs found in the lineages outside the Barnadesioideae. Taken together, these results show the enzymatic capacities of GAS and GAO emerged prior to the subsequent radiation of STL-producing Asteraceae subfamilies.

Food Matrix Effects of Polyphenol Bioaccessibility from Almond Skin during Simulated Human Digestion.

The goal of the present study was to quantify the rate and extent of polyphenols released in the gastrointestinal tract (GIT) from natural (NS) and blanched (BS) almond skins. A dynamic gastric model of digestion which provides a realistic simulation of the human stomach was used. In order to establish the effect of a food matrix on polyphenols bioaccessibility, NS and BS were either digested in water (WT) or incorporated into home-made biscuits (HB), crisp-bread (CB) and full-fat milk (FM). Phenolic acids were the most bioaccessible class (68.5% release from NS and 64.7% from BS). WT increased the release of flavan-3-ols (p < 0.05) and flavonols (p < 0.05) from NS after gastric plus duodenal digestion, whereas CB and HB were better vehicles for BS. FM lowered the % recovery of polyphenols, the free total phenols and the antioxidant status in the digestion medium, indicating that phenolic compounds could bind protein present in the food matrix. The release of bioactives from almond skins could explain the beneficial effects associated with almond consumption.

Emergence of terpene cyclization in Artemisia annua.

The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Here we report the first discovery of an epistatic network of residues that controls the onset of terpene cyclization in Artemisia annua. We begin with amorpha-4,11-diene synthase (ADS) and (E)-ß-farnesene synthase (BFS), a pair of terpene synthases that produce cyclic or linear terpenes, respectively. A library of ~27,000 enzymes is generated by breeding combinations of natural amino-acid substitutions from the cyclic into the linear producer. We discover one dominant mutation is sufficient to activate cyclization, and together with two additional residues comprise a network of strongly epistatic interactions that activate, suppress or reactivate cyclization. Remarkably, this epistatic network of equivalent residues also controls cyclization in a BFS homologue from Citrus junos. Fitness landscape analysis of mutational trajectories provides quantitative insights into a major epoch in specialized metabolism.

Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases.

Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography-mass spectrometry (GC-MS). In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.•We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs.•We validate the method by direct comparison with established assays. The agreement of k cat/K M among methods makes this adaptation optimal for rapid evaluation of TPSs.•We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries.

Evaluation of gastric processing and duodenal digestion of starch in six cereal meals on the associated glycaemic response using an adult fasted dynamic gastric model.

PURPOSE: To identify the key parameters involved in cereal starch digestion and associated glycaemic response by the utilisation of a dynamic gastro-duodenal digestion model. METHODS: Potential plasma glucose loading curves for each meal were calculated and fitted to an exponential function. The area under the curve (AUC) from 0 to 120 min and total digestible starch was used to calculate an in vitro glycaemic index (GI) value normalised against white bread. Microscopy was additionally used to examine cereal samples collected in vitro at different stages of gastric and duodenal digestion. RESULTS: Where in vivo GI data were available (4 out of 6 cereal meals) no significant difference was observed between these values and the corresponding calculated in vitro GI value. CONCLUSION: It is possible to simulate an in vivo glycaemic response for cereals when the gastric emptying rate (duodenal loading) and kinetics of digestible starch hydrolysis in the duodenum are known.

Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system.

Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5mg recombinant hGL per gram of infiltrated leaf tissue were obtained. Proteins expressed from two hGL constructs, wild type (wt-hGL) and with a Histidine tag at its C terminal (hGL-His), were purified from leaf tissue using Immobilized Lectin Affinity chromatography and Immobilized Metal Affinity chromatography. Both variants were glycosylated, enzymatically active, and had an apparent molecular weight similar to native hGL (approx. 50kDa). The recombinant hGLs were stable under acidic conditions and in the presence of gastric pepsin. Moreover, as found with the naturally occurring enzyme, the activity of recombinant hGL on the short chain lipid, tributyrin, was higher than on long chain Intralipid 30% emulsion. The maximum specific activity measured on tributyrin was 310 U/mg of protein and the maximum yield was 193 U/g of infiltrated leaf tissue. These results show that transient expression in plants can be used to produce active hGL that could be efficiently purified using established techniques. The approach provides a means of generating large quantities of hGL that could be of use for a number of applications both in vitro and in vivo.

Evaluation of the prebiotic properties of wheat arabinoxylan fractions and induction of hydrolase activity in gut microflora.

Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial bacteria such as lactobacilli and bifidobacteria in the human gastrointestinal tract. The effect of water-unextractable arabinoxylans (WU-AX) derived from wheat on the modulation of gut bacterial composition was investigated using a mixed culture fermentation system. A prebiotic index (PI) score of 2.03 was obtained after addition of 1% (w/v) WU-AX to a pH-controlled stirred anaerobic fermentation vessel. Pretreatment of the WU-AX with endo-beta-1,4-xylanase resulted in significantly higher PI value (3.48) indicating that pretreatment provided oligomers that were better utilised by the gut bacteria. The extracellular hydrolytic enzymes xylanase and ferulic acid esterase are both required for bacterial metabolism of WU-AX and both activities were present in supernatants derived from the mixed batch cultures. Addition of the WU-AX substrates to the batch cultures produced several fold increases of bacterial synthesis of both enzymes, and these increases were greater when the WU-AX substrate was pretreated with xylanase.

Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases.

Endo-beta1,4-xylanases (xylanases) hydrolyse the beta1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-alpha1,3-xylotriose (FAX(3)). The crystal structure of the EnXyn11A-FAX(3) complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites -3 and +2 allow accommodation of alpha1,2-linked 4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential -3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting beta strands 11 and 12, as suggested previously, but is highly complex.

In vitro three-stage continuous fermentation of wheat arabinoxylan fractions and induction of hydrolase activity by the gut microflora.

In vitro fermentations were carried out by using a model of the human colon to stimulate microbial activities of gut bacteria. The model consisted of a three-stage culture system. Bacterial populations were evaluated under the effect of three types of arabinoxylan, a nonstarch polysaccharide derived from wheat, the water-unextractable arabinoxylan fraction (WU-AX), WU-AX pretreated with exogenous xylanase and the soluble water-extractable arabinoxylan fraction (WE-AX). The xylanase pretreated (WU-AX) had a stimulatory effect upon colonic bifidobacteria throughout all three vessels. Counts of Bacteroides spp. and Clostridium spp. were also both significantly reduced. Addition of the WU-AX substrates to the first vessel resulted in induction of bacterial synthesis of extracellular hydrolytic enzymes xylanase and ferulic acid esterase which are both required for bacterial metabolism of WU-AX; this induction was significantly greater with the xylanase treated WU-AX.

Probing the structural basis for the difference in thermostability displayed by family 10 xylanases.

Thermostability is an important property of industrially significant hydrolytic enzymes: understanding the structural basis for this attribute will underpin the future biotechnological exploitation of these biocatalysts. The Cellvibrio family 10 (GH10) xylanases display considerable sequence identity but exhibit significant differences in thermostability; thus, these enzymes represent excellent models to examine the structural basis for the variation in stability displayed by these glycoside hydrolases. Here, we have subjected the intracellular Cellvibrio mixtus xylanase CmXyn10B to forced protein evolution. Error-prone PCR and selection identified a double mutant, A334V/G348D, which confers an increase in thermostability. The mutant has a Tm 8 degrees C higher than the wild-type enzyme and, at 55 degrees C, the first-order rate constant for thermal inactivation of A334V/G348D is 4.1 x 10(-4) min(-1), compared to a value of 1.6 x 10(-1) min(-1) for the wild-type enzyme. The introduction of the N to C-terminal disulphide bridge into A334V/G348D, which increases the thermostability of wild-type CmXyn10B, conferred a further approximately 2 degrees C increase in the Tm of the double mutant. The crystal structure of A334V/G348D showed that the introduction of Val334 fills a cavity within the hydrophobic core of the xylanase, increasing the number of van der Waals interactions with the surrounding aromatic residues, while O(delta1) of Asp348 makes an additional hydrogen bond with the amide of Gly344 and O(delta2) interacts with the arabinofuranose side-chain of the xylose moiety at the -2 subsite. To investigate the importance of xylan decorations in productive substrate binding, the activity of wild-type CmXyn10B, the mutant A334V/G348D, and several other GH10 xylanases against xylotriose and xylotriose containing an arabinofuranose side-chain (AX3) was assessed. The enzymes were more active against AX3 than xylotriose, providing evidence that the arabinose side-chain makes a generic contribution to substrate recognition by GH10 xylanases.

A family 10 Thermoascus aurantiacus xylanase utilizes arabinose decorations of xylan as significant substrate specificity determinants.

Xylan, which is a key component of the plant cell wall, consists of a backbone of beta-1,4-linked xylose residues that are decorated with arabinofuranose, acetyl, 4-O-methyl d-glucuronic acid and ferulate. The backbone of xylan is hydrolysed by endo-beta1,4-xylanases (xylanases); however, it is unclear whether the various side-chains of the polysaccharide are utilized by these enzymes as significant substrate specificity determinants. To address this question we have determined the crystal structure of a family 10 xylanase from Thermoascus aurantiacus, in complex with xylobiose containing an arabinofuranosyl-ferulate side-chain. We show that the distal glycone subsite of the enzyme makes extensive direct and indirect interactions with the arabinose side-chain, while the ferulate moiety is solvent-exposed. Consistent with the 3D structural data, the xylanase displays fourfold more activity against xylotriose in which the non-reducing moiety is linked to an arabinose side-chain, compared to the undecorated form of the oligosacchairde. These data indicate that the sugar decorations of xylans in the T.aurantiacus family 10 xylanase, rather than simply being accommodated, can be significant substrate specificity determinants.

Tailored catalysts for plant cell-wall degradation: redesigning the exo/endo preference of Cellvibrio japonicus arabinanase 43A.

Enzymes acting on polymeric substrates are frequently classified as exo or endo, reflecting their preference for, or ignorance of, polymer chain ends. Most biotechnological applications, especially in the field of polysaccharide degradation, require either endo- or exo-acting hydrolases, or they harness the essential synergy between these two modes of action. Here, we have used genomic data in tandem with structure to modify, radically, the chain-end specificity of the Cellvibrio japonicus exo-arabinanase CjArb43A. The structure of Bacillus subtilis endo-arabinanase 43A (BsArb43A) in harness with chain-end recognition kinetics of CjArb43A directed a rational design approach that led to the conversion of the Cellvibrio enzyme from an exo to an endo mode of action. One of the exo-acting mutants, D35L/Q316A, displays similar activity to WT CjArb43A and the removal of the steric block mediated by the side chains of Gln-316 and Asp-53 at the -3 subsite confers its capacity to attack internal glycoside bonds. This study provides a template for the production of tailored industrial catalysts. The introduction of subtle changes informed by comparative 3D structural and genomic data can lead to fundamental changes in the mode of action of these enzymes.

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan.

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.

Enzymic production of a feruloylated oligosaccharide with antioxidant activity from wheat flour arabinoxylan.

BACKGROUND: Main cereals such as rice, wheat, barley, and corn belong to the family Gramineae and have similar cell-wall composition. Since cereal cell walls are a good source of dietary fibre, meeting one-half of the daily requirement of 30 g of dietary fibre can be achieved by the regular consumption of cereals. Many studies have dealt with the isolation of feruloylated oligosaccharides from Gramineae by treatment with polysaccharide hydrolysing enzymes. AIM OF THIS STUDY: Therefore, the purpose of this study was to investigate the production of feruloylated oligosaccharides from insoluble wheat flour arabinoxylan (WFAX) by treatment with a Thermoascus aurantiacus family 10 endoxylanase (XYLI) and the evaluation of their antioxidant activity. METHODS: The main feruloylated oligosaccharide was purified by anion-exchange and size-exclusion chromatography (SEC). Alkaline saponification and acid hydrolysis were used for product identification. Evaluation of antioxidant activity was performed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction assay and the inhibition of copper-mediated oxidation of low density lipoprotein (LDL). RESULTS: The optimal conditions for WFAX hydrolysis using the XYLI have been determined to be 100 U g(-1) of WFAX for 30 min at 50 degrees C. Saponification of the oligosaccharide released FA and oligosaccharide. The released oligosaccharide consisted of arabinose and xylose in a molar ratio of 1:3 and these results support the identity of the feruloylated oligosaccharide as feruloyl arabinoxylotrisaccharide (FAX(3)). FAX(3) showed profound antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction assay exhibiting an antiradical efficiency of 0.035 (x 10(-3)) and inhibited the copper-mediated oxidation of human low density lipoprotein (LDL) in a dose-dependent manner with almost complete inhibition at 32 microM. CONCLUSION: A feruloylated oligosaccharide (FAX3) was isolated from WFAX after enzymatic treatment with XYLI. We verified antioxidant activity of FAX(3) which may be important in preventing or reducing the progression of atherosclerosis by inhibiting the peroxidation of lipoproteins.