A group of α-1,4-glucan lyase genes from the fungi Morchella costata,M. vulgaris and Peziza ostracoderma. Cloning,complete sequencing and heterologous expression

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, -1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121530 and 121971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.     

Cloning and characterization of pectate lyases expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus

Two pectate lyase genes (Bx-pel-1 and Bx-pel-2) were cloned from the pine wood nematode, Bursaphelenchus xylophilus. The deduced amino acid sequences of these pectate lyases are most similar to polysaccharide lyase family 3 proteins. Recombinant BxPEL1 showed highest activity on polygalacturonic acid and lower activity on more highly methylated pectin. Recombinant BxPEL1 demonstrated full dependency on Ca2+ for activity and optimal activity at 55 degrees C and pH 8 to 10 like other pectate lyases of polysaccharide lyase family 3. The protein sequences have predicted signal peptides at their N-termini and the genes are expressed solely in the esophageal gland cells of the nematode, indicating that the pectate lyases could be secreted into plant tissues to help feeding and migration in the tree. This study suggests that pectate lyases are widely distributed in plant-parasitic nematodes and play an important role in plant-nematode interactions.     

Cloning and Sequencing of Alginate Lyase Genes from Deep-Sea Strains of Vibrio and Agarivorans and Characterization of a New Vibrio Enzyme

Four alginate lyase genes were cloned and sequenced from the genomic DNAs of deep-sea bacteria, namely members of Vibrio and Agarivorans. Three of them were from Vibrio sp. JAM-A9m, which encoded alginate lyases, A9mT, A9mC, and A9mL. A9mT was composed of 286 amino acids and 57% homologous to AlxM of Photobacterium sp. A9mC (221 amino acids) and A9mL (522 amino acids) had the highest degree of similarity to two individual alginate lyases of Vibrio splendidus with 74% and 84% identity, respectively. The other gene for alginate lyase, A1mU, was shotgun cloned from Agarivorans sp. JAM-A1m. A1mU (286 amino acids) showed the highest homology to AlyVOA of Vibrio sp. with 76% identity. All alginate lyases belong to polysaccharide lyase family 7, although, they do not show significant similarity to one another with 14% to 58% identity. Among the above lyases, the recombinant A9mT was purified to homogeneity and characterized. The molecular mass of A9mT was around 28 kDa. The enzyme was remarkably salt activated and showed the highest thermal stability in the presence of NaCl. A9mT favorably degraded mannuronate polymer in alginate. We discussed substrate specificities of family 7 alginate lyases based on their conserved amino acid sequences.     

Genomic DNA structure of two new horseradish-peroxidase-encoding genes

Genomic DNAs encoding the horseradish peroxidase (HRP) isozymes, prxC2 and prxC3, were cloned and sequenced. By comparing the sequences of the HRP isozyme-encoding genes, prxC1a and prxC1b and their cDNA [Fujiyama et al., Eur. J. Biochem. 173 (1988) 681-687], , it was concluded that prxC2 and prxC3 consisted of four exons and three introns as in the prxC1 gene family. The position of introns in coding regions were the same in all four prx genes. Genes prxC2 and prxC3 coded for 347 and 349 amino acid (aa) residues, respectively, including putative signal sequences at the N termini. In the flanking regions of both genes, putative promoters and polyadenylation signals were found. Nucleotide sequence homology in the coding region was 71% between prxC1a and prxC2, and 66% between prxC1a and prxC3. The aa sequence homologies in plant and microbial peroxidases were compared.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of pectin lyase and pectinase sequences

A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.     

Peroxisomal isocitrate lyase of the n-alkane-assimilating yeast Candida tropicalis: gene analysis and characterization

A genomic DNA clone encoding isocitrate lyase, a key enzyme of the glyoxylate cycle and a peroxisomal enzyme of the n-alkane-assimilating yeast Candida tropicalis has been isolated with a cDNA probe from the yeast lambda EMBL library. Nucleotide sequence analysis of the genomic DNA clone disclosed that the region coding isocitrate lyase had a length of 1,650 base pairs, corresponding to 550 amino acids (61,602 Da). RNA blot analysis demonstrated that only one kind of mRNA (2 kb) supposed to be transcribed from this gene was present in the cells. A comparison of the amino acid sequences was made with the isocitrate lyase of castor bean, one of the glyoxysomal enzymes, and the enzyme of E. coli. The isocitrate lyases of C. tropicalis and castor bean had high homology, and the presence of some amino acid stretches conserved in all three enzymes suggests that these might be involved in the catalysis of the common reaction. There was an insertion common to the isocitrate lyases of C. tropicalis and castor bean, which is of interest concerning their evolution. In the C-terminal region, a characteristic sequence similar to that previously proposed as the import signal to peroxisomes was present.     

Cloning and Sequencing Analysis of Alginate Lyase Genes from the Marine Bacterium Vibrio sp. O2

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(β-D-mannuronate)lyases.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a non-coding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3′-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the ‘large’ sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species. This revised version was published online in November 2006 with corrections to the Cover Date.     

Sequencing and heterologous expression of an epimerase and two lyases from iminodisuccinate-degrading bacteria

Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes icl(B) and icl(S), encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the icl(B) gene was chromosomal like the corresponding icl(S) gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.     

The genes for the frog skin peptides GLa, xenopsin, levitide and caerulein contain a homologous export exon encoding a signal sequence and part of an amphiphilic peptide

From genomic libraries of Xenopus laevis, parts of the genes coding for the precursors of the skin peptides GLa (peptide with amino-terminal glycine and carboxy-terminal leucinamide), xenopsin and levitide have been isolated and sequenced. The gene for prepropeptide GLa comprises four exons, separated by relatively small introns. The gene for preproxenopsin is composed of five exons, of which all but the last one have been analyzed. This is a large gene encompassing at least 25,000 base pairs. In addition, two exons of the gene for preprolevitide have been isolated. A comparison of these genes reveals the presence of a homologous exon. This exon contains 161 bp, starts one base pair prior to the initiation codon and encodes a signal peptide and part of a pro region with processing sites. In addition, the two genes for preprocaerulein analyzed previously [Vlasak et al. (1987) Eur. J. Biochem. 169, 53-58] also contain a similar exon. This demonstrates the existence of a homologous export exon in genes encoding the precursors of different skin peptides.     

A new member of plant CS-lyases. A cystine lyase from Arabidopsis thaliana

Cystine lyases catalyze the breakdown of l-cystine to thiocysteine, pyruvate, and ammonia. Until now there are no reports of the identification of a plant cystine lyase at a molecular level, and it is not clear what biological role this class of enzymes have in plants. A cystine lyase was isolated from Brassica oleracea (L.), and partial amino acid sequencing allowed the corresponding full-length cDNA (BOCL3) to be cloned. The deduced amino acid sequence of BOCL3 showed highest homology to the deduced amino acid sequences of several Arabidopsis thaliana genes annotated as tyrosine aminotransferase-like, including a coronatine, jasmonic acid, and salt stress-inducible gene, CORI3 (78.8% identity), and the unidentified rooty/superroot1 gene (44.8% identity). A full-length expressed sequence tag clone of CORI3 was obtained and recombinant CORI3 was synthesized in Escherichia coli. Isolated recombinant CORI3 catalyzed a cystine lyase reaction, but no aminotransferase reactions. The present study identifies, for the first time, a cystine lyase from plants at a molecular level and redefines the functional assignment of the only functionally identified member of a group of A. thaliana genes annotated as tyrosine aminotransferase-like.     

Cloning and Sequence Analysis of Vibrio halioticoli Genes Encoding Three Types of Polyguluronate Lyase

The alginate lyase-coding genes of Vibrio halioticoli IAM 14596^T, which was isolated from the gut of the abalone Haliotis discus hannai, were cloned using plasmid vector pUC 18, and expressed in Escherichia coli. Three alginate lyase-positive clones, pVHB, pVHC, and pVHE, were obtained, and all clones expressed the enzyme activity specific for polyguluronate. Three genes, alyVG1, alyVG2, and alyVG3, encoding polyguluronate lyase were sequenced: alyVG1 from pVHB was composed of a 1056-bp open reading frame (ORF) encoding 352 amino acid residues; alyVG2 gene from pVHC was composed of a 993-bp ORF encoding 331 amino acid residues; and alyVG3 gene from pVHE was composed of a 705-bp ORF encoding 235 amino acid residues. Comparison of nucleotide and deduced amino acid sequences among AlyVG1, AlyVG2, and AlyVG3 revealed low homologies. The identity value between AlyVG1 and AlyVG2 was 18.7%, and that between AlyVG2 and AlyVG3 was 17.0%. A higher identity value (26.0%) was observed between AlyVG1 and AlyVG3. Sequence comparison among known polyguluronate lyases including AlyVG1, AlyVG2, and AlyVG3 also did not reveal an identical region in these sequences. However, AlyVG1 showed the highest identity value (36.2%) and the highest similarity (73.3%) to AlyA from Klebsiella pneumoniae. A consensus region comprising nine amino acid (YFKAGXYXQ) in the carboxy-terminal region previously reported by Mallisard and colleagues was observed only in AlyVG1 and AlyVG2. Received May 7, 1999; accepted September 4, 1999.     

Plant triose phosphate isomerase isozymes : purification, immunological and structural characterization, and partial amino Acid sequences

We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein.     

Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovora Er.

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for pectate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1% and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.     

Molecular identification of oligoalginate lyase of Sphingomonas sp. strain A1 as one of the enzymes required for complete depolymerization of alginate

A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37 degrees C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.     

Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics.

Three full-length cDNAs from alfalfa seedlings coding for hydroperoxide lyases were cloned and expressed in Escherichia coli and characterized as cytochrome P450 enzymes. The isoenzymes were specific for 13-hydroperoxy linoleic and linolenic acids and did not use the 9-hydroperoxy isomers as substrates. Because alfalfa contains both specificities, this indicates the presence of two different types of hydroperoxide lyases, each specific for one kind of substrate. The enzymes contain 480 amino acids (54 kDa) and contain an unusual, nonplastidic N-terminal sequence of 22 amino acids, which strongly reduces the enzyme activity. The only known presequence of a hydroperoxide lyase (from Arabidopsis thaliana) was considered to be a transit sequence. The reduced enzyme activity, however, indicates that the hydroperoxide lyases with N-terminal extensions could be pro-enzymes. This hypothesis is supported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased Vmax and Km compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have been obtained for characterization studies, by circumventing difficult purification procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned hydroperoxide lyases excellent tools to study the reaction mechanism and structure. We postulate an integrated reaction mechanism, based on the known chemistry of cytochrome P450 enzymes. This is the first mechanism that unifies all observed features of hydroperoxide lyases.