Enzymatic Strategy Research Articles

Frequency of Fabry disease in male and female haemodialysis patients in Spain

BackgroundFabry disease (FD), an X-linked lysosomal storage disorder, is caused by a reduced activity of the lysosomal enzyme α-galactosidase A. The disorder ultimately leads to organ damage (including renal failure) in males and females. However, heterozygous females usually present a milder phenotype with a later onset and a slower progression.MethodsA combined enzymatic and genetic strategy was used, measuring the activity of α-galactosidase A and genotyping the α-galactosidase A gene (GLA) in dried blood samples (DBS) of 911 patients undergoing haemodialysis in centers across Spain.ResultsGLA alterations were found in seven unrelated patients (4 males and 3 females). Two novel mutations (p.Gly346AlafsX347 and p.Val199GlyfsX203) were identified as well as a previously described mutation, R118C. The R118C mutation was present in 60% of unrelated patients with GLA causal mutations. The D313Y alteration, considered by some authors as a pseudo-deficiency allele, was also found in two out of seven patients.ConclusionsExcluding the controversial D313Y alteration, FD presents a frequency of one in 182 individuals (0.55%) within this population of males and females undergoing haemodialysis. Moreover, our findings suggest that a number of patients with unexplained and atypical symptoms of renal disease may have FD. Screening programmes for FD in populations of individuals presenting severe kidney dysfunction, cardiac alterations or cerebrovascular disease may lead to the diagnosis of FD in those patients, the study of their families and eventually the implementation of a specific therapy.

Intermediacy of Eudesmane Cation during Catalysis by Aristolochene Synthase

Aristolochene synthase from Penicillium roqueforti (PR-AS) catalyzes the formation of the bicyclic sesquiterpene (+)-aristolochene (5) from farnesyl diphosphate (1, FDP) in two mechanistically distinct cyclization reactions. The first reaction transforms farnesyl diphosphate to the uncharged intermediate (S)-(-)-germacrene A (3) through a macrocyclization process that links C1 and C10 upon magnesium ion-assisted diphosphate ester activation. In the second reaction mediated by PR-AS, a protonation induced cyclization has been suggested to generate the highly reactive trans-fused eudesmane cation 4 as a consequence of the precise folding of the enzyme-bound germacrene A intermediate. This contribution describes the use of the transition state analogue inhibitor 4-aza-eudesm-11-ene to explore the intermediacy of cation 4 as an on-path intermediate in the biosynthesis of aristolochene. 4-Aza-eudesm-11-ene as the hydrochloride salt 6 was stereospecifically synthesized in seven steps and 37% overall yield starting from chiral enamine 9. The synthetic sequence featured a highly regio- and stereoselective deracemization reaction of 9 that gave rise to the corresponding Michael adduct in >95% diastereomeric excess as evidenced by optical rotation and NMR measurements. 6 acts as a potent competitive inhibitor of PR-AS (K(i) = 0.35 +/- 0.12 microM) independent of the presence of diphosphate (K(i) = 0.24 +/- 0.09 microM). The failure of exogenous PP(i) to enhance the binding affinity of 6 for PR-AS could be interpreted against an eudesmyl cation/diphosphate anion pair mechanism as the enzymatic strategy to stabilize the highly reactive eudesmane cation 4. In addition, these observations seem to rule out simple favorable electrostatic and/or hydrogen bonding interactions between the active site anchored diphosphate ion and the ammonium ion 6 as the binding mode. Ammonium ion 6 seems to act as a genuine mimic of eudesmane cation (4) that most likely binds the active site of PR-AS in a productive conformation resembling that adapted by 4 during the PR-AS-catalyzed synthesis of 5.

Stereoselective enzymatic synthesis of monoglucosyl- myo-inositols with in vivo anti-inflammatory activity

The monoglucosyl-inositols α- d-glucopyranosyl-(1→4)-4 d- myo-inositol 3 and α- d-glucopyranosyl-(1→1)-1 d- myo-inositol 4 were synthesized by a combined enzymatic transglucosylation and hydrolysis strategy, using cyclodextrin glucosyl transferase (CGTase) from Thermoanaerobacter sp., followed by hydrolysis with Aspergillus niger glucoamylase. The glucosides were separated by preparative HPLC and fully characterized by extensive 1D and 2D NMR studies. The structure of the regioisomer 4 was confirmed by X-ray crystallography of its perbenzoylated derivative 4a. Both isomers demonstrated in vivo anti-inflammatory activity at comparative levels to corticosterone on mouse ear oedema induced by 12- O-tetradecanoylphorbol-13-acetate (TPA) and in rat hind paw oedema induced by carrageenan.

Trends for diverse production strategies of plant medicinal alkaloids

Covering: 1999 to 2009

Repair of DNA Alkylation Damage by the Escherichia coli Adaptive Response Protein AlkB as Studied by ESI‐TOF Mass Spectrometry

DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II)-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. In an effort to understand the range of acceptable substrates for AlkB, the enzyme was incubated with chemically synthesized oligonucleotides containing alkyl lesions, and the reaction products were analyzed by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. Consistent with the literature, but studied comparatively here for the first time, it was found that 1-methyladenine, 1,N 6-ethenoadenine, 3-methylcytosine, and 3-ethylcytosine were completely transformed by AlkB, while 1-methylguanine and 3-methylthymine were partially repaired. The repair intermediates (epoxide and possibly glycol) of 3,N 4-ethenocytosine are reported for the first time. It is also demonstrated that O 6-methylguanine and 5-methylcytosine are refractory to AlkB, lending support to the hypothesis that AlkB repairs only alkyl lesions attached to the nitrogen atoms of the nucleobase. ESI-TOF mass spectrometry is shown to be a sensitive and efficient tool for probing the comparative substrate specificities of DNA repair proteins in vitro.

Enzymatic and molecular strategies to diagnose Pompe disease

Enzyme replacement therapy for Pompe disease, a neuromuscular disorder characterized by lysosomal glycogen storage due to acid α-glucosidase deficiency, has entered the clinic. There is more than ever a need for early and reliable diagnosis. The objective of this review is to present a critical review of the recent literature on laboratory procedures to diagnose Pompe disease by enzymatic assay and DNA analysis. The methods we used were Compilation and expert interpretation of recent and relevant publications. The introduction of new and the updating of existing laboratory procedures have facilitated the diagnosis of Pompe disease (glycogen storage disease type II; acid maltase deficiency; OMIM 232300). With regard to enzymatic analysis, the application of acarbose as inhibitor of maltase-glucoamylase has enabled the use of mixed leukocyte preparations as diagnostic material. The use of glycogen as a natural substrate in the reaction mixture adds to the selectivity of this procedure. Newborn screening is envisaged and facilitated by the introduction of high-throughput procedures. DNA analysis has become an integral part of the diagnostic procedure for confirmation and completion, for carrier detection, and for genetic counseling.

ENZYMATIC SYNTHESIS OF COCOA BUTTER EQUIVALENT THROUGH TRANSESTERIFICATION OF PENTADESMA BUTYRACEA BUTTER

ABSTRACT A cocoa butter equivalent through enzymatic transesterification of Pentadesma butyracea butter with ethyl palmitate in an organic medium using immobilized lipase from Thermomyces lanuginosa was produced. The effects of several parameters such as initial ratio of ethyl palmitate – Triacylglycerols (TAGs) of P. butyracea, initial water activity of the enzyme preparation, solvent polarity and the enzyme loading were studied. The best result with regard to target TAGs was obtained in nonpolar solvents and low water activity. Thermograms of the products obtained by scanning differential calorimetry were similar to cocoa butter, but with minor differences, due particularly to the presence of trisaturated TAGs.PRACTICAL APPLICATIONSAn enzymatic strategy for the synthesis of cocoa butter obtained from an African butter from Pentadesma butyracea as starting material was devised. The product so obtained may be used as a cocoa butter substitute.

Bio-ethanol from water hyacinth biomass: An evaluation of enzymatic saccharification strategy

Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and β-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.

Enzymatic transglycosylation for glycoconjugate synthesis

Remarkable advances have been made in recent years in exploiting the transglycosylation activity of glycosidases and glycosynthase mutants for oligosaccharide and glycoconjugate synthesis. New glycosynthases were generated from retaining glycosidases, inverting glycosidases, and those that proceed in a mechanism of substrate-assisted catalysis. Directed evolution coupled with elegant screening methods has led to the discovery of an expanding number of glycosynthase mutants that show improved catalytic activity and/or altered substrate specificity. In particular, enzymatic transglycosylation strategy has been recently extended to the synthesis of complex glycoconjugates, including glycosphingolipids, N-glycoproteins, and other glycosylated natural products.

Juice clarification by protease and pectinase treatments indicates new roles of pectin and protein in cherry juice turbidity

Industrial juice clarification is accomplished by a combination of enzymatic depectinization, gelatin–silica sol, and/or bentonite treatment. The gelatin–silica sol treatment step is particularly slow, mischievous, and requires comprehensive downstream processing to obtain clarified juice. In this work, alternative, enzymatic clarification strategies of industrially pressed cherry juice were evaluated in a 2 4−1 factorial design set-up with addition of a pectinase, Pectinex Smash ®, a protease, Enzeco, both enzyme preparations derived from Aspergillus spp., gallic acid, and tannic acid as factors, and determination of turbidity, protein, pectin, and phenolics as responses. The effects of the alternative clarification treatments were assessed immediately after the particular clarification treatment (immediate turbidity) and during 14 days of cold storage (turbidity development). The protease treatment resulted in significant reduction of immediate turbidity, but had low clarification impact during the subsequent cold storage. In contrast, pectinase addition exerted a weak effect on immediate turbidity reduction, but effectively decreased the turbidity development during storage. The phenolic acid additions contributed to reduce turbidity when added together with the pectinase or the protease. However, when gallic acid and tannic acid were added together they induced enhanced turbidity formation. Conventionally, immediate turbidity is presumed to be caused by pectin, while turbidity development during cold storage (haze formation) is assumed to be due to protein–phenol interactions. Our results suggest that proteins play a decisive role in the formation of immediate turbidity in cherry juice, and point to that pectin may contribute to turbidity development during cold storage of cherry juice. The data may thus pave the way for development of improved, alternative procedures for cherry juice clarification.

Aromatic Residues in the Catalytic Center of Chitinase A from Serratia marcescens Affect Processivity, Enzyme Activity, and Biomass Converting Efficiency

The processive Serratia marcescens chitinases A (ChiA) and B (ChiB) are thought to degrade chitin in the opposite directions. A recent study of ChiB suggested that processivity is governed by aromatic residues in the +1 and +2 (aglycon) subsites close to the catalytic center. To further investigate the roles of aromatic residues in processivity and to gain insight into the structural basis of directionality, we have mutated Trp(167), Trp(275), and Phe(396) in the -3, +1, and +2 subsites of ChiA, respectively, and characterized the hydrolytic activities of the mutants toward beta-chitin and the soluble chitin-derivative chitosan. Although the W275A and F396A mutants showed only modest reductions in processivity, it was almost abolished by the W167A mutation. Thus, although aglycon subsites seem to steer processivity in ChiB, a glycon (-3) subsite seems to be adapted to do so in ChiA, in line with the notion that the two enzymes have different directionalities. Remarkably, whereas all three single mutants and the W167A/W275A double mutant showed reduced efficiency toward chitin, they showed up to 20-fold higher activities toward chitosan. These results show that the processive mechanism is essential for an efficient conversion of crystalline substrates but comes at a large cost in terms of intrinsic enzyme speed. This needs to be taken into account when devising enzymatic strategies for biomass turnover.

Acid-Catalyzed Oxygen-18 Labeling of Peptides

In enzymatic (18)O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as method for introducing (18)O label into peptides. Several synthetic peptides were dissolved in dilute acid containing 50% (v/v) H(2)(18)O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). (18)O-incorporation ratios were determined by linear regression analysis that allowed for multiple stable-isotope incorporations. At low pH, peptides exchanged their carboxyl oxygen atoms with the aqueous solvent. The isotope patterns gradually shifted to higher masses until they reached the expected binomial distribution at equilibrium after approximately 11 days. Reaction rates were residue- and sequence-specific. Due to its slow nature, the acid-catalyzed back exchange is expected to minimally interfere with enzymatic (18)O-labeling studies provided that storage and analysis conditions minimize low-pH exposure times. On its own, acid-catalyzed (18)O labeling is a general tagging strategy that is an alternative to the chemical, metabolic, and enzymatic isotope-labeling schemes currently used in quantitative proteomics.

Enzymatic deconstruction of xylan for biofuel production.

The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO(2)) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO(2) into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction.

A survey of chemical methods for sugar-nucleotide synthesis

Covering: up to 2009Sugar-nucleotides are essential intermediates in carbohydrate metabolism and glycoconjugate biosynthesis. Structurally, sugar-nucleotides are composed of a sugar or sugar derivative and a nucleoside mono- or diphosphate. Numerous combinations of sugars and nucleotides are found in nature, and although only nine different sugar-nucleotides have so far been identified in mammalian cells, this number is much greater in other organisms. Naturally occurring sugar-nucleotides, as well as structural analogues, are of great interest as substrates for enzymatic reactions in carbohydrate synthesis, as enzyme inhibitors, as tools for assay development and for the study of glycoconjugate biosynthesis. Therefore, methods for the efficient preparation of natural and non-natural sugar-nucleotides are of considerable importance for synthetic, biological and medicinal chemistry. The synthesis of sugar-nucleotides is non-trivial and complicated by a number of factors: the low solubility of sugar-nucleotides in organic solvents, the presence of several polar or charged functional groups, and the susceptibility of the glycosidic and pyrophosphate bonds to hydrolytic cleavage. To address these issues, both chemical and enzymatic strategies have been pursued. Here we present an overview of current methods for the chemical synthesis of NDP- and NMP-sugars, including recently developed protocols for the direct chemical manipulation of unprotected sugar-nucleotides.

Enantioselective enzymatic desymmetrization of prochiral 1,3-diols and enzymatic resolution of monoprotected 1,3-diols based on α-tetralone and related multifunctional scaffolds

Novel multifunctional chemotypes based on α-tetralone, α-indanone, and chromanone have been synthesized by a chemo-enzymatic approach by applying an enzymatic irreversible transesterification strategy. The scaffolds synthesized can be further elaborated with subsequent enzymatic as well as chemical transformations for the generation of new sets of structurally related organic molecules.

Water Treatment Residuals and Biosolids Co‐applications Affect Phosphatases in a Semi‐arid Rangeland Soil

Co‐application of biosolids and water treatment residuals (WTR) land has not been extensively studied but may be beneficial by sorbing excess biosolid‐borne or soil phosphorus (P) onto WTR, reducing the likelihood of off‐site movement. Reduction of excess soil P may affect the role of specific P‐cleaving enzymes. The research objective was to understand the long‐term effects of single co‐applications and the short‐term impacts of repeated co‐applications on soil acid phosphomonoesterase, phosphodiesterase, pyrophosphatase, and phytase enzyme activities. Test plots were 7.5 × 15 m with treatments consisting of three different WTR rates with a single biosolids rate (5, 10, and 21 Mg WTR ha−1; 10 Mg biosolids ha−1) surface co‐applied once in 1991 or reapplied in 2002. Control plots consisted of those that received no WTR–biosolids co‐applications and plots that received only 10 Mg biosolids ha−1. Plots were sampled to a 5‐cm depth in 2003 and 2004, and soil phosphatases and phytase enzyme activities were measured. Soil phosphodiesterase activity decreased in WTR‐amended plots, and pyrophosphatase activity decreased with increasing WTR application rates. In contrast, acid phosphatase and phytase activity increased with WTR addition, with WTR application possibly triggering a deficiency response causing microorganisms or plants to secrete these enzymes. Biosolids and WTR co‐applications may affect enzymatic strategies for P mineralization in this study site. Reductions in phosphodiesterase activity suggest less P mineralization from biomass sources, including nucleic acids and phospholipids. Increased acid phosphatase and phytase activities indicate that ester‐P and inositol‐P may be important plant‐available P sources in soils amended with WTR.

Biocatalytically Oligomerized Epicatechin with Potent and Specific Anti-proliferative Activity for Human Breast Cancer Cells

Catechins, naturally occurring flavonoids derived from wine and green tea, are known to exhibit multiple health benefits. Epigallocatechin gallate (EGCG) is one of the most widely investigated catechins, but its efficacy in cancer therapy is still inconsistent and limited. The poor stability of EGCG has contributed to the disparity in the reported anti-cancer activity and other beneficial properties. Here we report an innovative enzymatic strategy for the oligomerization of catechins (specifically epicatechin) that yields stable, water-soluble oligomerized epicatechins with enhanced and highly specific anti-proliferative activity for human breast cancer cells. This one-pot oxidative oligomerization is carried out in ambient conditions using Horseradish Peroxidase (HRP) as a catalyst yielding water-soluble oligo(epicatechins). The oligomerized epicatechins obtained exhibit excellent growth inhibitory effects against human breast cancer cells with greater specificity towards growth-inhibiting cancer cells as opposed to normal cells, achieving a high therapeutic differential. Our studies indicate that water-soluble oligomeric epicatechins surpass EGCG in stability, selectivity and efficacy at lower doses.

Systems- and Molecular-Level Elucidation of Signaling Processes Through Chemistry

Complete understanding of the molecular mechanisms of signal transduction is a major focus of research in the postgenomic era. Efforts to realize this goal have benefited from interdisciplinary approaches, specifically through the development of new technologies. Several groups have now described new chemical strategies to probe signaling with broadly different degrees of focus. The first report employs an enzymatic tagging strategy to monitor global proteolytic cleavage events that occur during apoptosis. The second study illustrates a general method to functionally characterize a specific kinase through the identification of its substrates. Finally, the third report describes a synthetic route to generate ubiquitinated proteins and demonstrates the utility of protein chemistry in understanding the detailed biochemical mechanisms of signaling. Together, these papers highlight the power of chemical tools to examine specific networks, thereby expanding our understanding of signaling pathways.

Isolation and Identification of Oligomers from Partial Degradation of Lime Fruit Cutin

Complementary degradative treatments with low-temperature hydrofluoric acid and methanolic potassium hydroxide have been used to investigate the protective biopolymer cutin from Citrus aurantifolia (lime) fruits, augmenting prior enzymatic and chemical strategies to yield a more comprehensive view of its molecular architecture. Analysis of the resulting soluble oligomeric fragments with one- and two-dimensional NMR and MS methods identified a new dimer and three trimeric esters of primary alcohols based on 10,16-dihydroxyhexadecanoic acid and 10-oxo-16-hydroxyhexadecanoic acid units. Whereas only 10-oxo-16-hydroxyhexadecanoic acid units were found in the oligomers from hydrofluoric acid treatments, the dimer and trimer products isolated to date using diverse degradative methods included six of the seven possible stoichiometric ratios of monomer units. A novel glucoside-linked hydroxyfatty acid tetramer was also identified provisionally, suggesting that the cutin biopolymer can be bound covalently to the plant cell wall. Although the current findings suggest that the predominant molecular architecture of this protective polymer in lime fruits involves esters of primary and secondary alcohols based on long-chain hydroxyfatty acids, the possibility of additional cross-linking to enhance structural integrity is underscored by these and related findings of nonstandard cutin molecular architectures.

Expression of NAD+ dependent 15‐hydroxyprostaglandin dehydrogenase and protection of prostaglandins in human hair follicle

NAD(+) dependent 15-hydroxyprostaglandin dehydrogenate (15-PGDH) catalyses oxidation of 15(S)-hydroxyl group of prostaglandins and as a result inactivates their physiological potential. Positive effects of prostaglandins or prostaglandin analogues were reported on terminal hair, vellus hair or eyelash growth and a complex prostaglandin network was recently described in human hair follicle. In the present study, we showed that 15-PGDH was expressed in human hair follicle mainly in melanocytes and keratinocytes, which brought us to consider this enzyme as a possible target to sustain local prostaglandin production. Using a recombinant enzymatic strategy, specific 15-PGDH inhibitors were screened. We identified a thiazolidine dione derivative exhibiting efficacy on follicular outer root sheath keratinocytes, since it concomitantly decreased the production of deactivated 13,14 dihydro 15-ketoprostaglandin F(2alpha) and sustained prostaglandin F(2alpha)in vitro production. In the context of recent interest in prostaglandins and prostaglandin analogues as hair regrowth agents, we postulated that the use of selected 15-PGDH inhibitors could reinforce or prolong the effect of these physiological mediators on hair and skin.