Production of recombinant cholesterol oxidase containing covalently bound FAD in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO).     

Overexpression,one-step purification,and characterization of a type II cholesterol oxidase from a local isolate <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. PTCC 1633

A 1.6 kb gene encoding a cholesterol oxidase (choR) from a local isolate, Rhodococcus sp. PTCC 1633 was cloned into pET23a and the highly expressed recombinant enzyme was purified from the cell lysate of IPTG-induced Escherichia coli BL21(DE3)pLysS with one-step absorption on cholesterol. The purified protein had a molecular mass of 55 kDa, isoelectric point at about pH 9.0 and absorption peaks at 280, 380 and 460 nm, indicating that the enzyme is a flavoprotein. The optimum pH and temperature for the recombinant enzyme were 7.0 and 50°C, respectively. Steady-state kinetic revealed that the cholesterol oxidase had a K _m of 32 μM. This study is the first report concerning expression and one-step purification of a gene encoding cholesterol oxidase from Rhodococcus spp. This study revealed that this enzyme is a type II cholesterol oxidase.     

Molybdenum hydroxylases in Drosophila. III. Further characterization of the low xanthine dehydrogenase gene

The biochemical effects of several newly induced low xanthine dehydrogenase (lxd) mutations in Drosophila melanogaster were investigated. When homozygous, all lxd alleles simultaneously interrupt each of the molybdoenzyme activities to approximately the same levels: xanthine dehydrogenase, 25%; aldehyde oxidase, 12%; pyridoxal oxidase, 0%; and sulfite oxidase, 2% as compared to the wild type. In order to evaluate potentially small complementation or dosage effects, mutant stains were made coisogenic for 3R. These enzymes require a molybdenum cofactor, and lxd cofactor levels are also reduced to less than 10% of the wild type. These low levels of molybdoenzyme activities and cofactor activity are maintained throughout development from late larval to adult stages. The lxd alleles exhibit a dosage-dependent effect on molybdoenzyme activities, indicating that these mutants are leaky for wild-type function. In addition, cofactor activity is dependent upon the number of lxd ⁺ genes present. The lxd mutation results in the production of more thermolabile XDH and AO enzyme activities, but this thermolability is not transferred with the cofactor to a reconstituted Neurospora molybdoenzyme. The lxd gene is localized to salivary region 68 A4-9, 0.1 map unit distal to the superoxide dismutase (Sod) gene.     

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.     

Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I,from Bacillus subtilis – Bsu121I,a type II restriction endonuclease from Bacillus subtilis

A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified. The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease. The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp. The endonuclease activity required Mg⁺² as cofactor like other type II endonucleases.     

The presence of two serine racemases in Streptomyces garyphalus,a d-cycloserine producer

Two serine racemases (I and II) were isolated from Streptomyces garyphalus. Serine racemase I (molecular weight 93,000) was purified to a single band in an analytical electrofocusing system. Serine racemase II (molecular weight 73,000) was partially purified. Both enzymes used pyridoxal-5-phosphate as cofactor. Besides serine the enzymes utilized alanine as substrate but no other amino acid tested. The K _m values of l-alanine and l-serine for enzyme I were 111 mM and 35 mM respectively. Enzyme I was not inhibited by d-cycloserine but by hydroxylamine. Both substances inhibited enzyme II. The serine racemases may be involved in the biosynthesis of d-cycloserine in S. garyphalus.     

Molybdenum hydroxylases in Drosophila

Summary The molybdenum hydroxylases are a ubiquitous class of enzymes which contain molybdenum in association with a low molecular weight cofactor. Genetic evidence suggests that the Drosophila loci, ma-1, cin and lxd are concerned with this cofactor because mutants for any one of these loci simultaneously interrupt activity for two molybdenum hydroxylases, XDH and A0. A third enzyme activity, P0, is also absent in each of the three mutants but evidence classifying P0 as a molybdoenzyme has been lacking. This study utilizes the known tungsten sensitivity of molybdoenzymes to demonstrate directly that pyridoxal oxidase is also a molybdoenzyme. The low molecular weight molybdenum cofactor is found to be severely reduced in extracts of the lxd and cin mutants but ma-1 mutants have high levels of cofactor. A partially purified preparation of XDH crossreacting material from ma-1 was also shown to contain the molybdenum cofactor. These results, considered with data from other workers are taken to indicate that the functions of all three of the loci examined could be concerned with some aspect of cofactor biosynthesis.This work was supported by PHS grant GM 23736 to V. Finnerty     

Coenzyme precursor-assisted expression of a cholesterol oxidase from <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene (choB _b ), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs and encodes a protein of 551 amino acids. ChoB _b exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia coli BL21–CodonPlus (DE3)-RP grown at 23°C in Luria-Bertani medium containing 50 μM riboflavin, the precursor of the FAD coenzyme of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choB_b.     

Purification and Some Properties of α-Galactosidase from Candida guilliermondii H-404

Candida guilliermondii H-404, isolated from soil, produced thermostable α-galactosidase, but small amounts of other glycosidases (such as β-galactosidase, α-glucosidase, and β-glucosidase). The enzyme was separated into two fractions by DEAE-Toyopearl 650M chromatography, and the two enzymes were designated galactosidase I and II. These two enzymes had the same molecular weight (270,000 by gel filtration, 64,000 by SDS-PAGE). The isoelectric points of α-galactosidase I and II were 6.16 and 6.21, respectively. These two enzymes were different from each other in pH stability, temperature stability, and effects of Fe^{2 +} and Cu^{2 +} ion on α-galactosidase activity. The enzyme had stronger transfer activity and wider acceptor specificity than α-galactosidases which have been reported.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

Molecular Cloning of a Genomic DNA Encoding Yam Class IV Chitinase

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.     

Properties of protease-treated cytochrome c oxidase from beef heart

Summary About 45% of the protein can be removed from oxidized cytochrome c oxidase by treatment with proteolytic enzymes under a variety of conditions, leading to an increased heme to protein ratio.The principal spectroscopic parameters of cytochrome c oxidase are retained in the protease-treated enzyme.Of the overall catalytic activity 20% remained after digestion; the electron-transfer reactions were impaired but the affinity for cytochrome c appeared unchanged.Proteolysis resulted in removal of the hydrophobic subunit III and most of the smaller hydrophilic subunits, leaving a core, which basically consists of the two largest subunits I and II.The subunits I and/or II carry the prosthetic groups of the enzyme and at least one of the cytochrome c binding sites. The smaller subunits, however, are essential for optimal electron transfer and possibly have other functions as well.     

Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2

Rhodococcus sp. R_14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH₂-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a rather wide pH range of 4.0–10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly oxidizes 3β-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3α-hydroxysteroids. Thus, the presence of a 3β-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated at 55 μM; the COX activity was markedly inhibited by metal ions such as Hg²⁺ and Fe³⁺ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione. These suggests that -SH groups may be involved in the catalytic activity of the present COX.     

Investigation of Cu(I)-dependent 2,4,5-trihydroxyphenylalanine quinone biogenesis in Hansenula polymorpha amine oxidase

Copper, a mediator of redox chemistries in biology, is often found in enzymes that bind and reduce dioxygen. Among these, the copper amine oxidases catalyze the oxidative deamination of primary amines utilizing a type(II) copper center and 2,4,5-trihydroxyphenylalanine quinone (TPQ), a covalent cofactor derived from the post-translational modification of an active site tyrosine. Previous studies established the dependence of TPQ biogenesis on Cu(II); however, the dependence of cofactor formation on the biologically relevant Cu(I) ion has remained untested. In this study, we demonstrate that the apoform of the Hansenula polymorpha amine oxidase readily binds Cu(I) under anaerobic conditions and produces the quinone cofactor at a rate of 0.28 h(-1) upon subsequent aeration to yield a mature enzyme with kinetic properties identical to the protein product of the Cu(II)-dependent reaction. Because of the change in magnetic properties associated with the oxidation of copper, electron paramagnetic resonance spectroscopy was employed to investigate the nature of the rate-limiting step of Cu(I)-dependent cofactor biogenesis. Upon aeration of the unprocessed enzyme prebound with Cu(I), an axial Cu(II) electron paramagnetic resonance signal was found to appear at a rate equivalent to that for the cofactor. These data provide strong evidence for a rate-limiting release of superoxide from a Cu(II)(O(2)(.)) complex as a prerequisite for the activation of the precursor tyrosine and its transformation for TPQ. As copper is trafficked to intracellular protein targets in the reduced, Cu(I) state, these studies offer possible clues as to the physiological significance of the acquisition of Cu(I) by nascent H. polymorpha amine oxidase.     

Cyanide as a copper and quinone-directed inhibitor of amine oxidases from pea seedlings (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and<Emphasis Type="Italic"> Arthrobacter globiformis</Emphasis>: evidence for both copper coordination and cyanohydrin derivatization of the quinone cofactor

The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques. Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ_amr-Cu(II)TPQ_sq-Cu(I). The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O₂ for reoxidation of TPQ. Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs. This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ. K_d values for Cu(II)-CN^– and Cu(I)-CN^–, as well as the K_i for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO. In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN^– complexation of Cu(I).Abbreviations AGAO Arthrobacter globiformis amine oxidase - APAO Arthrobacter P1 amine oxidase - APT attached proton test - BPAO bovine plasma amine oxidase - CuAO quinone-copper containing amine oxidase - LTQ lysyl tyrosylquinone - MAO monoamine oxidase - PKAO porcine kidney amine oxidase - PPAO porcine plasma amine oxidase - PSAO pea seedling amine oxidase - TPQ 2,4,5-trihydroxyphenylalaninequinone - TPQ_amr TPQ aminoresorcinol - TPQ_imq TPQ iminoquinone - TPQ_ox TPQ oxidized - TPQ_sq TPQ semiquinone - WT wild-typeE.M. Shepard and G.A. Juda contributed equally to this workThis revised version was published online in February 2004: Hansenula polymorpha was not italicised at the end of the Introduction, Equation 3 appeared twice, and the resolution of Scheme 3 was insufficient.An erratum to this article can be found at     

Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

Summary Mutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamon's female sterility is also discussed.     

Cloning of amadoriase I isoenzyme from Aspergillus sp.: evidence of FAD covalently linked to Cys342

Amadoriases are a novel class of FAD enzymes which catalyze the oxidative deglycation of glycated amino acids to yield corresponding amino acids, glucosone, and H(2)O(2). We previously reported the purification and characterization of two amadoriase isoenzymes from Aspergillus fumigatus and the molecular cloning of amadoriase II. To identify the primary structure of amadoriase I, we prepared a cDNA library from Aspergillus fumigatus and isolated a clone using a probe amplified by polymerase chain reaction with primers designed according to the partial amino acid sequences from peptide mapping. The primary structure of the enzyme deduced from the nucleotide sequence comprises 445 amino acid residues. The enzyme contains 1 mol of FAD as a cofactor, which is covalently linked to Cys342, as determined by mutagenesis analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and electrospray ionization-collisional-activated dissociation tandem mass spectrometry. Sequence alignment studies show that amadoriase I has 22% homology with monomeric sarcosine oxidase in which FAD is also linked to a homologous Cys residue. Amadoriases are of potential importance as tools for uncoupling hyperglycemia and glycation reactions that are thought to play a role in diabetic complications.     

Protein lysine 6-oxidase (lysyl oxidase) cofactor: methoxatin (PQQ) or pyridoxal?

1. Treatment of chick embryos with two lathyrogens lowered lysyl oxidase and increased collagen extractability. 2. Subsequent treatment with pyridoxal restored both parameters towards normal, whereas PQQ treatment was less effective. 3. These results suggest the requirement of a pyridoxal derivative for the formation of the enzyme, acting either as cofactor or because its formation requires some pyridoxal-dependent enzyme. The cochromatography of the enzyme with [3H]pyridoxine-derived radioactivity supports the cofactor role. 4. The conclusions of other authors that lysyl oxidase contains PQQ relates to enzymes from other species or to amine oxidases not characterised as lysyl oxidase.     

Studies on (Na + K)-activated ATPase XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation

(1) The neutral lipids and the free and bound fatty acids of a highly purified (Na⁺ + K⁺)-ATPase preparation from rabbit kidney outer medulla have been analysed. (2) On a dry weight basis, the total lipid content is nearly the same as the total protein content, and consists for 66% of phospholipids and for 34% of neutral lipids and free fatty acids. In the latter category cholesterol is the main component (71%). (3) On a molar basis the enzyme preparation contains 382 mol phospholipids, 67 mol free fatty acids, 9, 16 and 12 mol mono-, di- and triacylglycerols, 249 and 19 mol free and esterified cholesterol per mol enzyme. (4) The fatty acid composition of each lipid and of the free fatty acid fraction, present in the enzyme preparation, is reported. (5) All cholesterol and part of the phospholipids can be removed by hexane extraction, leaving 66% of the (Na⁺ + K⁺)-ATPase activity. Oxidation of all cholesterol to cholest-4-en-3-one by cholesterol oxidase leaves 85% of the (Na⁺ + K⁺)-ATPase activity. These results indicate that cholesterol is not essential for (Na⁺ + K⁺)-ATPase activity.