The feruloyl esterase system of Talaromyces stipitatus: determining the hydrolytic and synthetic specificity of TsFaeC

The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was probed using a series of C1-C4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of k(cat) and k(cat)/K(m) values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water-organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.     

Biochemical characteristics of three feruloyl esterases with a broad substrate spectrum from Bacillus amyloliquefaciens H47

Feruloyl esterases (Faes) are a subclass of the carboxylic esterases that hydrolyze the ester bonds between ferulic acid and polysaccharides in plant cell walls. Until now, the biochemical characteristics of FAEs from Bacillus spp. have not been reported. In this study, a strain with high activity of FAEs, Bacillus amyloliquefaciens H47 was screened from 122 Bacillus – type strains. Finally, three FAEs (BaFae04, BaFae06, and BaFae09) were identified. Comparing with other bacterial FAEs, these novel FAEs exhibited low sequence identities (less than 30%). The profiles of 52 esterase substrates showed that the three FAEs had a broad substrate spectrum and could effectively hydrolyze several common FAE substrates, such as methyl ferulate, ethyl caffeate, methyl p-coumarate, methyl sinapate, and chlorogenic acid. Furthermore, the three FAEs also can release ferulic acid from destarched wheat bran. They showed maximal activity with an optimal pH of 8.0 at 30 °C, 35 °C, and 40 °C, respectively. BaFae04 showed high stability in the temperature range of 25–60 °C for 1 h and retained 59% of its activity at 60 °C. The present study displays some useful characteristics of FAEs for potential industrial application and contributes to our understanding of FAEs.     

Production and characterization of the Talaromyces stipitatus feruloyl esterase FAEC in Pichia pastoris: identification of the nucleophilic serine

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. We report the over-expression and characterization of a novel feruloyl esterase exhibiting broad substrate specificity from Talaromyces stipitatus (FAEC) in Pichia pastoris. Using various gene constructions, we have investigated the use of alternative signal peptides to produce an authentic feruloyl esterase featuring the N-terminal sequence determined for the native enzyme. We demonstrate that additional amino acids at the N-terminus of the FAEC sequence do not influence the catalytic capacity of the enzyme, and that the nature of the signal sequence has a limited effect on the yield of the secreted enzyme, with the T. stipitatus FAEC signal sequence producing 297 mgL(-1), the Neurospora crassa Fae-1 260 mgL(-1), and the Saccharomyces cerevisiae alpha-factor secretion signal 214 mgL(-1). Mature FAEC contains two internal peptide sequences that correspond with the consensus motif G-X-S-X-G that contains the catalytic serine nucleophile, which is conserved in the esterase enzyme superfamily. The serine residues at the center of these peptide motifs have been independently mutated and the corresponding enzymes have been over-expressed in P. pastoris to identify the candidate nucleophilic residue responsible for catalyzing the enzymatic reaction. Purified recombinant FAEC containing S465A retained the esterase activity and appeared unaffected by the amino acid modification. In contrast, FAEC activity containing S166A was below the HPLC detection limit, suggesting that serine 166 constitutes the nucleophile.     

Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger

Feruloyl esterases hydrolyse phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure making material more accessible to glycoside hydrolases. Here we describe the crystal structure of inactive S133A mutant of type-A feruloyl esterase from Aspergillus niger (AnFaeA) in complex with a feruloylated trisaccharide substrate. Only the ferulic acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH(3) groups with the hydroxyl groups of Tyr80. The importance of aromatic and polar residues in the activity of AnFaeA was also evaluated using site-directed mutagenesis. Four mutant proteins were heterologously expressed in Pichia pastoris, and their kinetic properties determined against methyl esters of ferulic, sinapic, caffeic and p-coumaric acid. The k(cat) of Y80S, Y80V, W260S and W260V was drastically reduced compared to that of the wild-type enzyme. However, the replacement of Tyr80 and Trp260 with smaller residues broadened the substrate specificity of the enzyme, allowing the hydrolysis of methyl caffeate. The role of Tyr80 and Trp260 in AnFaeA are discussed in light of the three-dimensional structure.     

Comparison of mesophilic and thermophilic feruloyl esterases: characterization of their substrate specificity for methyl phenylalkanoates

The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using methyl esters of phenylalkanoic acids. Only 13 out of 26 substrates tested were significant substrates for all the enzymes. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Maintaining the phenylpropanoate structure but altering the substitutions of the aromatic ring demonstrated that the type-A esterase from the mesophilic fungus Fusarium oxysporum (FoFaeA) showed a preference for methoxylated substrates, in contrast to the type-B esterase from the same source (FoFaeB) and the thermophilic type-B (StFaeB) and type-C (StFaeC) from Sporotrichum thermophile, which preferred hydroxylated substrates. All four esterases hydrolyzed short chain aliphatic acid (C2-C4) esters of p-nitrophenol, but not the C12 ester of laurate. All the feruloyl esterases were able to release ferulic acid from the plant cell wall material in conjunction with a xylanase, but only the type-A esterase FoFaeA was effective in releasing the 5,5' form of diferulic acid. The thermophilic type-B esterase had a lower catalytic efficiency than its mesophilic counterpart, but released more ferulic acid from plant cell walls.     

Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae2) was identified as a type C feruloyl esterase. The RuFae2 alone released ferulic acid from rice bran, wheat bran, wheat-insoluble arabinoxylan, corn fiber, switchgrass, and corn bran in the order of decreasing activity. Using a saturating amount of RuFae2 for 100 mg substrate, a maximum of 18.7 and 80.0 μg FA was released from 100 mg corn fiber and wheat-insoluble arabinoxylan, respectively. Addition of GH10 endoxylanase (EX) synergistically increased the release of FA with the highest level of 6.7-fold for wheat bran. The synergistic effect of adding GH11 EX was significantly smaller with all the substrates tested. The difference in the effect of the two EXs was further analyzed by comparing the rate in the release of FA with increasing EX concentration using wheat-insoluble arabinoxylan as the substrate.     

Murine hexose-6-phosphate dehydrogenase: a bifunctional enzyme with broad substrate specificity and 6-phosphogluconolactonase activity

Murine hexose-6-phosphate dehydrogenase has been purified from liver microsomes by affinity chromatography on 2('),5(')-ADP-Sepharose. The purified enzyme has 6-phosphogluconolactonase activity and glucose-6-phosphate dehydrogenase activity and has a native molecular mass of 178 kDa and a subunit molecular mass of 89 kDa. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucosamine 6-phosphate, and glucose 6-sulfate are substrates for murine hexose-6-phosphate dehydrogenase, with either NADP or deamino-NADP as coenzyme. This study confirms that hexose-6-phosphate dehydrogenase is a bifunctional enzyme which can catalyze the first two reactions of the pentose phosphate pathway.     

The Arabidopsis AtSTE24 is a CAAX protease with broad substrate specificity

Following prenylation, the proteins are subject to two prenyl-dependent modifications at their C-terminal end, which are required for their subcellular targeting. First, the three C-terminal residues of the CAAX box prenylation signaling motif are removed, which is followed by methylation of the free carboxyl group of the prenyl cysteine moiety. An Arabidopsis homologue of the yeast CAAX protease STE24 (AFC1) was cloned and expressed in rce1 Delta ste24 Delta mutant yeast to demonstrate functional complementation. The petunia calmodulin CaM53 is a prenylated protein terminating in a CTIL CAAX box. Coupled methylation proteolysis assays demonstrated the processing of CaM53 by AtSTE24. In addition, AtSTE24 promoted plasma membrane association of the GFP-Rac fusion protein, which terminates with a CLLM CAAX box. Interestingly, a plant homologue of the second and major CAAX protease in yeast and animal cells, RCE1, was not identified despite the availability of vast amounts of sequence data. Taken together, these data suggest that AtSTE24 may process several prenylated proteins in plant cells, unlike its yeast homologue, which processes only a-mating factor, and its mammalian homologue, for which prenyl-CAAX substrates have not been established. Transient expression of GFPAtSTE24 in leaf epidermal cells of Nicotiana benthamiana showed that AtSTE24 is exclusively localized in the endoplasmic reticulum, suggesting that prenylated proteins in plants are first targeted to the endoplasmic reticulum following their prenylation.     

Molecular cloning, overexpression and characterization of a novel feruloyl esterase from a soil metagenomic library

The gene estF27, encoding a protein with feruloyl esterase activity, was cloned through functional screening from a soil metagenomic library and expressed in Escherichiacoli BL21 (DE3) with high solubility. Sequence analysis showed that estF27 encoded a protein of 291 amino acids with a predicted molecular mass of 31.16 kDa. According to the substrate specificity, EstF27 was classified as a type A feruloyl esterase. EstF27 displayed optimal activity at 40°C and pH 6.8. This enzyme was stable in a broad pH range of 5.0-10.0 over 24 h, and retained more than 50% of its activity after 96 or 120 h incubation in the presence of 3 M KCl or 5 M NaCl. The enzyme activity was slightly enhanced by the addition of Mg(2+) and Fe(3+) at a low concentration, and completely inhibited by Cu(2+). In the enzymatic hydrolysis of destarched wheat bran, EstF27 could release ferulic acid from it in the presence of xylanase from Thermomyces lanuginosus. Given its alkalitolerance, halotolerance and highly soluble expression, EstF27 is a promising candidate for industrial applications.     

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite(Coptotermes formosanus) gut

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and 37oC, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.     

Moraxella catarrhalis Lgt2, a galactosyltransferase with broad acceptor substrate specificity

The genetic basis of lipo-oligosaccharide (LOS) biosynthesis for the bacterium Moraxella catarrhalis has been elucidated and functions suggested for each of the glycosyltransferases. In this study we have expressed and characterised one of these enzymes, the putative galactosyltransferase Lgt2_B/C. The lgt2_B/C gene was amplified from M. catarrhalis, expressed in Escherichia coli, and Lgt2_B/C was purified. Analysis of its glycosyltransferase catalytic activity ascertained the pH and temperature optima. The donor specificity and acceptor specificity were examined and they showed that Lgt2_B/C is a galactosyltransferase with relatively broad acceptor specificity with optimal activity in the presence of exogenous Mg²⁺.     

Cloning,expression, and characterization of a human 4'-phosphopantetheinyl transferase with broad substrate specificity

A single candidate 4'-phosphopantetheine transferase, identified by BLAST searches of the human genome sequence data base, has been cloned, expressed, and characterized. The human enzyme, which is expressed mainly in the cytosolic compartment in a wide range of tissues, is a 329-residue, monomeric protein. The enzyme is capable of transferring the 4'-phosphopantetheine moiety of coenzyme A to a conserved serine residue in both the acyl carrier protein domain of the human cytosolic multifunctional fatty acid synthase and the acyl carrier protein associated independently with human mitochondria. The human 4'-phosphopantetheine transferase is also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes. The same human protein also has recently been implicated in phosphopantetheinylation of the alpha-aminoadipate semialdehyde dehydrogenase involved in lysine catabolism (Praphanphoj, V., Sacksteder, K. A., Gould, S. J., Thomas, G. H., and Geraghty, M. T. (2001) Mol. Genet. Metab. 72, 336-342). Thus, in contrast to yeast, which utilizes separate 4'-phosphopantetheine transferases to service each of three different carrier protein substrates, humans appear to utilize a single, broad specificity enzyme for all posttranslational 4'-phosphopantetheinylation reactions.     

The carotenoid cleavage dioxygenase 1 enzyme has broad substrate specificity, cleaving multiple carotenoids at two different bond positions

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9',10') bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9',10') bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5',6' bond positions of lycopene but not geranylgeranyl diphosphate, zeta-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7' and 8') and carbons 11 and 12 (11' and 12') as well as the methyl groups on carbons 5, 9, and 13 (5', 9', and 13').     

The study of substrate specificity of a new peptide substrate of endothelin-converting enzyme

A new hexapeptide CMC-Ala-Gly-Gly-Trp-Phe-Arg was used as a substrate for assay of endothelin-converting (ECE; EC 3.4.24.71) and angiotensin-converting (ACE; EC 3.4.15.1) enzymes and of neutral endopeptidase (NEP; EC 3.4.24.11). The specific inhibitors lisinopril (for ACE) and thiorphan (for NEP) were used for discrimination between activities of these enzymes.     

Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme

The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS. Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T. cruzi. Both recombinant and authentic T. cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds. Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors. Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated. TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate. The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T. cruzi TAT. Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Ialpha aspartate and aromatic aminotransferases is critical for T. cruzi's TAT activity.     

Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay

Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarstr?m, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.     

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.