Cloning and expression of fructosyl-amino acid oxidase gene from Corynebacterium sp. 2-4-1 in Escherichia coli

The gene encoding the fructosyl-amino acid oxidase (fructosyl-alpha-L-amino acid: oxygen oxidoreductase (defructosylating); EC 1.5.3) of Corynebacterium sp. 2-4-1 was cloned and expressed in Escherichia coli. The gene consists of 1,116 nucleotides and encodes a protein of 372 amino acids with a predicted molecular mass of 39,042. The open reading frame was confirmed as the gene of the fructosyl-amino acid oxidase by comparison with the N-terminal amino acid sequence of the purified fructosyl-amino acid oxidase from Corynebacterium sp. 2-4-1. The sequence of the AMP-binding motif, GXGXXG, was found in the deduced N-terminal region. The amino acid sequence of the fructosyl-amino acid oxidase showed no similarity to that of fungal fructosyl-amino acid oxidases. In addition, substrate specificities of this fructosyl-amino acid oxidase were different from those of other fructosyl-amino acid oxidases. The fructosyl-amino acid oxidase of Corynebacterium sp. 2-4-1 is an enzyme that has unique substrate specificity and primary structure in comparison with fungal fructosyl-amino acid oxidases.     

Cloning and sequencing of two Ceriporiopsis subvermispora bicupin oxalate oxidase allelic isoforms: implications for the reaction specificity of oxalate oxidases and decarboxylases

Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess < or =0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.     

Cloning,sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.     

Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.     

Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200

Burkholderia cepacia strain ST-200 produces an extracellular cholesterol oxidase which is stable and highly active in the presence of organic solvents. This cholesterol oxidase produces 6beta-hydroperoxycholest-4-en-3-one from cholesterol, with the consumption of two moles of O2 and the formation of one mole of H2O2. The structural gene encoding the cholesterol oxidase was cloned and sequenced. The primary translation product was predicted to be 582 amino acid residues. The mature product is composed of 539 amino acid residues and is preceded by a signal sequence of 43 residues. The cloned gene was expressed as an active product in Escherichia coli and the product was localized in the periplasmic space. The cholesterol oxidase produced from E. coli was purified to homogeneity from the periplasmic fraction. The purified enzyme was highly stable in the presence of various organic solvents or detergents, as compared with the commercially available cholesterol oxidases tested.     

Cloning,sequence analysis,and expression of a gene encoding <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K _m and V _max values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The V _max/K _m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.     

Nucleotide sequence of the gene for monoamine oxidase (maoA) from Escherichia coli

We found that the structural gene for monoamine oxidase was located at 30.9 min on the Escherichia coli chromosome. Deletion analysis showed that two amine oxidase genes are located in this region. The nucleotide sequence of one of the two genes was determined. The peptide sequence of the first 40 amino acids from the N terminus of monoamine oxidase purified from E. coli agrees with that deduced from the nucleotide sequence of the gene. The leader peptide extends over 30 amino acids. The nucleotide sequence of the gene and amino acid sequence of the predicted mature enzyme (M.W. 81,295) were highly homologous to those of the maoA_K gene and monoamine oxidase from Klebsiella aerogenes, respectively. From these results and analysis of the enzyme activity, we concluded that the gene encodes for monoamine oxidase (maoA_E). The tyrosyl residue, which may be converted to topa quinone in the E. coli enzyme, was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidases.     

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.     

Structural insights into sulfite oxidase deficiency

Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.     

Comparative study of soluble and immobilized phenol oxidase from the fungus Mycelia sterilia IBR 35219/2

Phenol oxidase (EC 1.14.18.1) from the microscopic fungus Mycelia sterilia IBR 35219/2 was immobilized using glutaraldehyde on macroporous silica carriers. The enzyme immobilized on amino-Silochrome SKh-2 or aminopropyl-Silochrome 350/80 exhibited maximum activity. Soluble and immobilized phenol oxidases were compared. Compared to the soluble enzyme, the activity of which was optimum at pH 5.5, immobilized phenol oxidase exhibited optimum activity under slightly more acidic conditions (pH 5.2). Immobilization considerably increased the enzyme stability. Both soluble and immobilized forms of phenol oxidase from M. sterilia IBR 35219/2 catalyze oxidative conversion of phenolic compounds of the green tea extract.     

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.     

The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases

The cytochrome o complex is one of two ubiquinol oxidases in the aerobic respiratory system of Escherichia coli. This enzyme catalyzes the two-electron oxidation of ubiquinol-8 which is located in the cytoplasmic membrane, and the four-electron reduction of molecular oxygen to water. The purified oxidase contains at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and has been shown to couple electron flux to the generation of a proton motive force across the membrane. In this paper, the DNA sequence of the cyo operon, containing the structural genes for the oxidase, is reported. This operon is shown to encode five open reading frames, cyoABCDE. The gene products of three of these, cyoA, cyoB, and cyoC, are clearly related to subunits II, I, and III, respectively, of the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. This family of cytochrome c oxidases contain heme a and copper as prosthetic groups, whereas the E. coli enzyme contains heme b (protoheme IX) and copper. The most striking sequence similarities relate the large subunits (I) of both the E. coli quinol oxidase and the cytochrome c oxidases. It is likely that the sequence similarities reflect a common molecular architecture of the two heme binding sites and of a copper binding site in these enzymes. In addition, the cyoE open reading frame is closely related to a gene denoted ORF1 from Paracoccus dentrificans which is located in between the genes encoding subunits II and III of the cytochrome c oxidase of this organism. The function of the ORF1 gene product is not known. These sequence relationships define a superfamily of membrane-bound respiratory oxidases which share structural features but which have different functions. The E. coli cytochrome o complex oxidizes ubiquinol but has no ability to catalyze the oxidation of reduced cytochrome c. Nevertheless, it is clear that the E. coli oxidase and the aa3-type cytochrome c oxidases must have very similar structures, at least in the vicinity of the catalytic centers, and they are very likely to have similar mechanisms for bioenergetic coupling (proton pumping).     

Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus.     

Soluble and immobilized phenol oxidase of the fungusMycelia sterilia IBR 35219/2: A comparative study

Phenol oxidase (EC 1.14.18.1) from the microscopic fungusMycelia sterilia IBR 35219/2 was immobilized using glutaraldehyde on macroporous silica carriers. The enzyme immobilized on amino-Silochrome SKh-2 or aminopropyl-Silochrome 350/80 exhibited maximum activity. Soluble and immobilized phenol oxidases were compared. Compared to the soluble enzyme, the activity of which was optimum at pH 5.5, immobilized phenol oxidase exhibited optimum activity under slightly more acidic conditions (pH 5.2). Immobilization considerably increased enzyme stability. Both soluble and immobilized forms of phenol oxidase fromM. sterilia IBR 35 219/2 catalyze oxidative conversion of phenolic compounds of green tea extract.     

D-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes.

D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.     

cDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a cDNA encoding the peroxisomal trifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase (HDE) from the yeast Candida tropicalis pK233. Poly(A)+RNA isolated from C. tropicalis cells grown in oleic acid medium was used to construct a cDNA library in lambda gt11. The library was screened with a polyclonal antiserum against HDE. A recombinant was confirmed to encode HDE by hybridization-selection translation and immunoprecipitation. The HDE cDNA (HDE) has a single open reading frame of 2718 nt, encoding a protein of 905 amino acids, not including the initiator methionine. The Mr of the protein is 99,350. A partial gene duplication is believed to have occurred in the evolution of the HDE gene. Codon utilization in the gene is not random, with 86.0% of the amino acids specified by 23 preferentially used codons, a situation similar to that found in genes encoding peroxisomal catalase and the various fatty acyl-CoA oxidases from C. tropicalis. The increase in HDE activity in C. tropicalis cells grown in oleic acid medium as opposed to glucose medium is due, at least in part, to increased HDE-specific mRNA levels.     

Isolation and expression of the gene encoding yeast mitochondrial malate dehydrogenase.

The mitochondrial tricarboxylic acid cycle enzyme malate dehydrogenase was purified from Saccharomyces cerevisiae, and an antibody to the purified enzyme was obtained in rabbits. Immunoscreening of a yeast genomic DNA library cloned into a lambda gt11 expression vector with anti-malate dehydrogenase immunoglobulin G resulted in identification of a lambda recombinant encoding an immunoreactive beta-galactosidase fusion protein. The yeast DNA portion of the coding region for the fusion protein translates into an amino acid sequence which is very similar to carboxy-terminal sequences of malate dehydrogenases from other organisms. In s. cerevisiae transformed with a multicopy plasmid carrying the complete malate dehydrogenase gene, the specific activity and immunoreactivity of the mitochondrial isozyme are increased by eightfold. Expression of both the chromosomal and plasmid-borne genes is repressed by growth on glucose. Disruption of the chromosomal malate dehydrogenase gene in haploid S. cerevisiae produces mutants unable to grow on acetate and impaired in growth on glycerol plus lactate as carbon sources.     

Discovery of a gene involved in a third bacterial protoporphyrinogen oxidase activity through comparative genomic analysis and functional complementation

Tetrapyrroles are ubiquitous molecules in nearly all living organisms. Heme, an iron-containing tetrapyrrole, is widely distributed in nature, including most characterized aerobic and facultative bacteria. A large majority of bacteria that contain heme possess the ability to synthesize it. Despite this capability and the fact that the biosynthetic pathway has been well studied, enzymes catalyzing at least three steps have remained "missing" in many bacteria. In the current work, we have employed comparative genomics via the SEED genomic platform, coupled with experimental verification utilizing Acinetobacter baylyi ADP1, to identify one of the missing enzymes, a new protoporphyrinogen oxidase, the penultimate enzyme in heme biosynthesis. COG1981 was identified by genomic analysis as a candidate protein family for the missing enzyme in bacteria that lacked HemG or HemY, two known protoporphyrinogen oxidases. The predicted amino acid sequence of COG1981 is unlike those of the known enzymes HemG and HemY, but in some genomes, the gene encoding it is found neighboring other heme biosynthetic genes. When the COG1981 gene was deleted from the genome of A. baylyi, a bacterium that lacks both hemG and hemY, the organism became auxotrophic for heme. Cultures accumulated porphyrin intermediates, and crude cell extracts lacked protoporphyrinogen oxidase activity. The heme auxotrophy was rescued by the presence of a plasmid-borne protoporphyrinogen oxidase gene from a number of different organisms, such as hemG from Escherichia coli, hemY from Myxococcus xanthus, or the human gene for protoporphyrinogen oxidase.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.