Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune

The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-d-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.     

Synthetic esters recognized by glucuronoyl esterase from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Glucuronoyl esterase is a novel carbohydrate esterase recently discovered in the cellulolytic system of the wood-rotting fungus Schizophyllum commune on the basis of its ability to hydrolyze methyl ester of 4-O-methyl-d-glucuronic acid. This substrate was not fully corresponding to the anticipated function of the enzyme to hydrolyze esters between xylan-bound 4-O-methyl-d-glucuronic acid and lignin alcohols occurring in plant cell walls. In this work we showed that the enzyme was capable of hydrolyzing two synthetic compounds that mimic the ester linkages described in lignin-carbohydrate complexes, esters of 4-O-methyl-d-glucuronic and d-glucuronic acid with 3-(4-methoxyphenyl)propyl alcohol. A comparison of kinetics of hydrolysis of methyl and 3-(4-methoxyphenyl)propyl esters indicated that the glucuronoyl esterase recognizes the uronic acid part of the substrates better than the alcohol type. The catalytic efficiency of the enzyme was much higher with the ester of 4-O-methyl-d-glucuronic acid than with that of d-glucuronic acid. Examination of the action of glucuronoyl esterase on a series of methyl esters of 4-O-methyl-d-glucopyranuronosyl residues α-1,2-linked to xylose and several xylooligosaccharides suggested that the rate of deesterification is independent of the character of the carbohydrate part glycosylated by the 4-O-methyl-d-glucuronic acid.     

Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene

At least three acetyl xylan esterases (AXE I, II and III) are secreted by Penicillium purpurogenum. This publication describes more detailed work on AXE I and its gene. AXE I binds cellulose but not xylan; it is glycosylated and inactivated by phenylmethylsulphonyl fluoride, showing that it is a serine esterase. The axe1 gene presents an open reading frame of 1278 bp, including two introns of 68 and 61 bp; it codes for a signal peptide of 31 residues and a mature protein of 351 amino acids (molecular weight 36,693). AXE I has a modular structure: a catalytic module at the amino terminus belonging to family 1 of the carbohydrate esterases, a linker rich in serines and threonines, and a family 1 carboxy terminal carbohydrate binding module (CBM). The CBM is similar to that of AXE from Trichoderma reesei, (with a family 5 catalytic module) indicating that the genes for catalytic modules and CBMs have evolved separately, and that they have been linked by gene fusion. The promoter sequence of axe1 contains several putative sequences for binding of gene expression regulators also found in other family 1 esterase gene promoters. It is proposed that AXE I and II act in succession in xylan degradation; first, xylan is attacked by AXE I and other xylanases possessing CBMs (which facilitate binding to lignocellulose), followed by other enzymes acting mainly on soluble substrates.     

Identification of genes encoding microbial glucuronoyl esterases

One type of covalent linkages connecting lignin and hemicellulose in plant cell walls is the ester linkage between 4-O-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. An enzyme that could hydrolyze such linkages, named glucuronoyl esterase, occurs in the cellulolytic system of the wood-rotting fungus Schizophyllum commune. Here we report partial amino acid sequences of the enzyme and the results of subsequent search for homologous genes in sequenced genomes. The homologous genes of unknown functions were found in genomes of several filamentous fungi and one bacterium. The gene corresponding to the cip2 gene of Hypocrea jecorina (Trichoderma reesei), known to be up-regulated under conditions of induction of cellulolytic and hemicellulolytic enzymes, was over-expressed in H. jecorina. The product of the cip2 gene was purified to homogeneity and shown to exhibit glucuronoyl esterase activity.     

Amplification and methylation of an esterase gene associated with insecticide-resistance in greenbugs, Schizaphis graminum (Rondani) (Homoptera: Aphididae)

The greenbug aphid, Schizaphis graminum (Rondani) has developed resistance to organophosphorus insecticides by the over-production of esterases that have been classified as Type I and Type II. The first twenty N-terminal amino acids of the Type I esterase were determined and used to design an oligonucleotide, which in conjunction with an active site primer derived from conserved sequences of other insect esterases and two internal primers specific for esterases from another aphid species resulted in a 0.85 kb genomic DNA fragment from resistant greenbugs. This was extended by 5′ RACE which provided approximately 1.2 kb of the 5′ end of the esterase gene. The 5′ DNA sequence corresponded to 19 of the 20 known amino acids of the Type I esterase, with the last needing only a one base change (probably resulting from a PCR artifact). Furthermore, the sequence showed very close similarity to the amplified E4/FE4 esterase genes of Myzus persicae (Sulzer). A comparison of sequences suggested that the S. graminum gene has introns in the same positions as the first two introns of E4/FE4, with the second intron being considerably larger in S. graminum. Probing of Southern blots with the 0.85 kb esterase fragment showed that the gene encoding the Type I esterase is amplified 4- to 8-fold in resistant S. graminum and that the amplified sequences contain 5-methylcytosine at MspI/HpaII sites, again in agreement with previous findings for M. persicae genes.     

ISOLATION AND SEQUENCE ANALYSIS OF A cDNA ENCODING A NOVEL PUTATIVE ESTERASE FROM THE MARINE MICROALGA ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE,HAPTOPHYTA)1

Microalgae constitute an interesting novel study area for characterizing new esterases, and so we decided to isolate a complete cDNA encoding a new putative microalgal esterase from the haptophyte Isochrysis galbana Parke. Rapid amplifications of both the 5′ and 3′ cDNA ends (RACE) were performed with specific primers, designed using an incomplete candidate gene from the I. galbana expressed sequence tag (EST) database. The full‐length cDNA obtained was designated IgEst1. The coding sequence was 828 bp long, and the deduced amino acid sequence revealed a polypeptide of 275 amino acids with a predicted signal peptide of 23 residues in the N‐terminal region. The following 252 amino acids formed, after in silico analysis, a mature protein with a molecular mass of ～26.92 kDa and had a theoretical pI of 5.87. Alignment analyses revealed slight but significant identity and similarity with carboxylesterases, phospholipases, and lysophospholipases from various organisms including fungi, plants, and animals. The new sequence IgEst1 enclosed the catalytic triad Ser/Asp/His and the consensus pentapeptide Gly‐X‐Ser‐X‐Gly, two highly conserved patterns found in serine hydrolases. Phylogenetic analyses established a close relationship with putative esterases identified in microalgae genomes.     

Molecular characterization of two novel esterase genes from carmine spider mite,Tetranychus cinnabarinus (Acarina: Tetranychidae)

Abstract Two novel esterase complementary DNAs were identified and cloned from the insecticide-susceptible strain of Tetranychus cinnabarinus (Boisduval) (Acarina: Tetranychidae), which were designated as TCE1 and TCE2, respectively. The cDNA of TCE1 gene contained an open reading frame (ORF) of 1701 bp encoding 567 amino acids, and a predicted molecular weight of 62.75 kDa, the cDNA of TCE2 contained an ORF of 1680 bp encoding 560 amino acids, and a predicted molecular weight of 63.14 kDa. TCE1 and TCE2 were submitted to GenBank, accession number EU130461 and EU130462. The well-conserved sequence motif, GXSXG, used as a signature pattern in the esterase family are present in both TCE1 and TCE2 (GQSAG in TCE1, whereas GESAG in TCE2), indicating that these two genes are predicted to be esterases. Comparison of the deduced amino acid sequence with the published mite esterase sequence coming from Boophilus microplus showed that TCE1 shares 33.98% identity and TCE2 shares 33.46% identity. TCE1 and TCE2 share 46.4% identity. Quantitative real-time polymerase chain reaction revealed that expression level of the TCE2 gene was relatively higher than that of the TCE1 in all instars examined except the protonymph, and the expression level of these two esterase genes in adults of T. cinnabarinus was significantly higher than that in any other instars, respectively. T. cinnabarinus is an important agricultural mite pest and esterases are important in the metabolisms of insects and mites; the genomic information obtained in this study will contribute to esterase molecular biological study on mite pest species.     

Hyperthermostable acetyl xylan esterase

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90°C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl⁻¹ in the absence of substrate was about 13 h at 98°C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100–104°C and 107.5°C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.     

Nonspecific esterases of Mus musculus

Seventeen genes controlling the expression of carboxylic ester hydrolases, commonly known as esterases, have been identified in the mouse Mus musculus. Seven esterase loci are found on chromosome 8, where two clusters of esterase loci occur. It seems probable that the genes within these clusters have arisen from a common ancestral gene by tandem duplication. Close linkage of esterase genes is also found in the rat, rabbit, and prairie vole. Some mouse esterases appear to be homologous with certain human esterases. The function of these nonspecific enzymes is still unknown.     

An analysis of molecular polymorphism of esterase M produced by motileAeromonas strains

The high levels of electrophoretic polymorphism of esterase M detected in eight distinct hybridization groups of motileAeromonas raise questions of genetic homogeneity of the electromorphs. The 40 electromorphs detected fall in fourM _r classes—75, 80, 90, and 110 kD—and one typical variant belonging to each of these classes was purified. The four purified esterases exhibited the same resistance to heat, topH and to diisopropyl fluorophosphate, the sameK _m values for 1-naphthyl acetate and 1-naphthyl propionate (1mm), and immunological cross-reactions. Within each class, the electromorphs appeared to be related in term of single amino acid substitutions as estimated from their comparative titration patterns. The titration curves of the four purified esterases were strictly parallel suggesting close structural similarities. Thus, despite considerable variation in theirpI,M _F,andM _r values, it seems likely that the variants of esterase M are the products of closely related loci originating from a common ancestral gene.This work was supported by a grant from the Conseil Scientifique de la Faculté Xavier Bichat (Université Paris VII).     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

D genome doners for Aegilops cylindrica (CCDD) and Triticum aestivum (AABBDD) deduced from esterase isozyme analysis

Summary Putative D genome donors for Aegilops cylindrica (2n = 28, CCDD) and Triticum aestivum (2n = 42, AABBDD) were studied with the isoelectric focusing patterns of esterase isozymes. 103 strains of Ae. cylindrica were uniform in their isozyme pattern. 30 strains of the putative parent, Ae. caudata, showed no zymogram variation, whereas the other parent, Ae. squarrosa, comprised 3 phenotypes. Natural Ae. cylindrica had an isozyme pattern which corresponded to a mixture of esterases from Ae. caudata and type 3 Ae. squarrosa. Therefore, it is concluded that the D genome donor of Ae. cylindrica is derived from type 3 Ae. squarrosa. These results suggest that Ae. cylindrica originated with a single amphiploidy event, and the C and D genomes have remained remarkably constant regarding esterase isozyme composition.On the other hand, T. aestivum comprised three zymogram phenotypes. These phenotypes contain bands which can be ascribed to the D genome of type 2 Ae. squarrosa. These results suggest that the D genome of Ae. cylindrica differs from that of T. aestivum. Evolution of the AB and D genomes of T. aestivum is indicated by the zymogram polymorphism. The origin of Ae. cylindrica is possibly more recent than that of T. aestivum.Contribution No. 433 of the Laboratory of Genetics, Faculty of Agriculture, Kyoto University     

Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when α‐naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub‐cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)‐derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional‐genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut. © 2009 Wiley Periodicals, Inc.     

Inheritance of esterases in the white crappie (Pomoxis annularis), black crappie (P. nigromaculatus), and their F1 and F2 interspecific hybrids*

White crappie, Pomoxis annularis, and black crappie, P. nigromaculatus, were hybridized to produce Fi and F2 generations. There was considerable tissue specific esterase gene function within each parental species. Many of the esterases were homologous but possessed different electrophoretic mobilities between the two species which permitted their use as gene markers. The pattern of inheritance and expression of a number of esterase genes was determined in the Fi and F2 interspecific hybrids. Although certain key morphological phenotypes characteristic of the black crappie predominate in the Fi hybrid, the enzyme phenotypes reflect the contributions of both co-dominant parental genomes. There is a higher percentage of heterozygotes than expected in the F2 generation which may be indicative of heterosis. The genetic analysis also provided a better understanding of the polypeptide composition of these esterases.     

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure

A family 15 carbohydrate esterase (CE15) from the white‐rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col‐0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl‐4‐O‐methyl‐d ‐glucopyranuronate and could target ester linkages that contribute to lignin–carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf‐yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT‐IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross‐linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross‐linking within plant cell walls.     

Proteome Composition in Plasmodium falciparum: Higher Usage of GC-Rich Nonsynonymous Codons in Highly Expressed Genes

The parasite Plasmodium falciparum, responsible for the most deadly form of human malaria, is one of the extremely AT-rich genomes sequenced so far and known to possess many atypical characteristics. Using multivariate statistical approaches, the present study analyzes the amino acid usage pattern in 5038 annotated protein-coding sequences in P. falciparum clone 3D7. The amino acid composition of individual proteins, though dominated by the directional mutational pressure, exhibits wide variation across the proteome. The Asn content, expression level, mean molecular weight, hydropathy, and aromaticity are found to be the major sources of variation in amino acid usage. At all stages of development, frequencies of residues encoded by GC-rich codons such as Gly, Ala, Arg, and Pro increase significantly in the products of the highly expressed genes. Investigation of nucleotide substitution patterns in P. falciparum and other Plasmodium species reveals that the nonsynonymous sites of highly expressed genes are more conserved than those of the lowly expressed ones, though for synonymous sites, the reverse is true. The highly expressed genes are, therefore, expected to be closer to their putative ancestral state in amino acid composition, and a plausible reason for their sequences being GC-rich at nonsynonymous codon positions could be that their ancestral state was less AT-biased. Negative correlation of the expression level of proteins with respective molecular weights supports the notion that P. falciparum, in spite of its intracellular parasitic lifestyle, follows the principle of cost minimization. [Reviewing Editor : Dr. Richard Kliman]     

Structural and biochemical characterization of a metagenome‐derived esterase with a long N‐terminal extension

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC‐Est1～6, were isolated from a fosmid library of a leaf‐branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome‐derived enzymes, LC‐Est1 is characterized by the presence of a long N‐terminal extension (LNTE, residues 26–283) between a putative signal peptide (residues 1–25) and a C‐terminal esterase domain (residues 284–510). A putative esterase from Candidatus Solibacter usitatus (CSu‐Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC‐Est1. To examine whether LC‐Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC‐Est1 (residues 26–510), LC‐Est1C (residues 284–510), and LC‐Est1C* (residues 304–510) were overproduced in E. coli, purified, and characterized. LC‐Est1C* was only used for structural analysis. The crystal structure of LC‐Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC‐Est1C was lower than that of LC‐Est1 by 60%, although its substrate specificity was similar to that of LC‐Est1. LC‐Est1C was less stable than LC‐Est1 by 3.3°C. These results suggest that LNTE of LC‐Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.     

Analysis of the <Emphasis Type="Italic">MAT1-2-1</Emphasis> gene of <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.