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(1/1596) Human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames.

A universal molybdenum-containing cofactor (MoCo) is essential for the activity of all human molybdoenzymes, including sulphite oxidase. The free cofactor is highly unstable, and all organisms share a similar biosynthetic pathway. The involved enzymes exhibit homologies, even between bacteria and humans. We have exploited these homologies to isolate a cDNA for the heterodimeric molybdopterin (MPT)-synthase. This enzyme is necessary for the conversion of an unstable precursor into molybdopterin, the organic moiety of MoCo. The corresponding transcript shows a bicistronic structure, encoding the small and large subunits of the MPT-synthase in two different open reading frames (ORFs) that overlap by 77 nucleotides. In various human tissues, only one size of mRNA coinciding with the bicistronic transcript was detected. In vitro translation and mutagenesis experiments demonstrated that each ORF is translated independently, leading to the synthesis of a 10-kDa protein and a 21-kDa protein for the small and large subunits, respectively, and indicated that the 3'-proximal ORF of the bicistronic transcript is translated by leaky scanning.  (+info)

(2/1596) Human molybdopterin synthase gene: genomic structure and mutations in molybdenum cofactor deficiency type B.

Biosynthesis of the molybdenum cofactor (MoCo) can be divided into (1) the formation of a precursor and (2) the latter's subsequent conversion, by molybdopterin synthase, into the organic moiety of MoCo. These two steps are reflected by the complementation groups A and B and the two formally distinguished types of MoCo deficiency that have an identical phenotype. Both types of MoCo deficiency result in a pleiotropic loss of all molybdoenzyme activities and cause severe neurological damage. MOCS1 is defective in patients with group A deficiency and has been shown to encode two enzymes for early synthesis via a bicistronic transcript with two consecutive open reading frames (ORFs). MOCS2 encodes the small and large subunits of molybdopterin synthase via a single transcript with two overlapping reading frames. This gene was mapped to 5q and comprises seven exons. The coding sequence and all splice site-junction sequences were screened for mutations, in MoCo-deficient patients in whom a previous search for MOCS1 mutations had been negative. In seven of the eight patients whom we investigated, we identified MOCS2 mutations that, by their nature, are most likely responsible for the deficiency. Three different frameshift mutations were observed, with one of them found on 7 of 14 identified alleles. Furthermore, a start-codon mutation and a missense mutation of a highly conserved amino acid residue were found. The locations of the mutations confirm the functional role of both ORFs. One of the patients with identified MOCS2 mutations had been classified as type B, in complementation studies. These findings support the hypothetical mechanism, for both forms of MoCo deficiency, that formerly had been established by cell-culture experiments.  (+info)

(3/1596) Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity.

The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.  (+info)

(4/1596) Biochemical characterization and solution structure of nitrous oxide reductase from Alcaligenes xylosoxidans (NCIMB 11015).

Nitrous oxide reductase (N2OR) is the terminal enzyme involved in denitrification by microbes. No three-dimensional structural information has been published for this enzyme. We have isolated and characterised N2OR from Alcaligenes xylosoxidans (AxN2OR) as a homodimer of M(r) 134,000 containing seven to eight copper atoms per dimer. Comparison of sequence and compositional data with other N2ORs suggests that AxN2OR is typical and can be expected to have similar domain folding and subunit structure to other members of this family of enzymes. We present synchrotron X-ray-scattering data, analysed using a model-independent method for shape restoration, which gave a approximately 20 A resolution structure of the enzyme in solution, providing a glimpse of the structure of any N2OR and shedding light on the molecular architecture of the molecule. The specific activity of AxN2OR was approximately 6 mumol of N2O reduced.min-1. (mg of protein)-1; N2OR activity showed both base and temperature activation. The visible spectrum exhibited an absorption maximum at 550 nm with a shoulder at 635 nm. On oxidation with K3Fe(CN)6, the absorption maximum shifted to 540 nm and a new shoulder at 480 nm appeared. Reduction under anaerobic conditions resulted in the formation of an inactive blue form of the enzyme with a broad absorption maximum at 650 nm. As isolated, the enzyme shows an almost featureless EPR spectrum, which changes on oxidation to give an almost completely resolved seven-line hyperfine signal in the gII region, g = 2.18, with AII = 40 G, consistent with the enzyme being partially reduced as isolated. Both the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a CuA centre.  (+info)

(5/1596) The strict molybdate-dependence of glucose-degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme--an aldehyde oxidoreductase.

In order to investigate the effects of trace elements on different metabolic pathways, the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been cultivated on various carbon substrates in the presence and absence of molybdate. When grown on glucose (but neither on glutamate nor casein hydrolysate) as sole carbon source, the lack of molybdate results in serious growth inhibition. By analysing cytosolic fractions of glucose adapted cells for molybdenum containing compounds, an aldehyde oxidoreductase was detected that is present in the cytosol to at least 0.4% of the soluble protein. With Cl2Ind (2,6-dichlorophenolindophenol) as artificial electron acceptor, the enzyme exhibits oxidizing activity towards glyceraldehyde, glyceraldehyde-3-phosphate, isobutyraldehyde, formaldehyde, acetaldehyde and propionaldehyde. At its pH-optimum (6.7), close to the intracellular pH of Sulfolobus, the glyceraldehyde-oxidizing activity is predominant. The protein has an apparent molecular mass of 177 kDa and consists of three subunits of 80.5 kDa (alpha), 32 kDa (beta) and 19.5 kDa (gamma). It contains close to one Mo, four Fe, four acid-labile sulphides and four phosphates per protein molecule. Methanol extraction revealed the existence of 1 FAD per molecule and 1 molybdopterin per molecule, which was identified as molybdopterin guanine dinucleotide on the basis of perchloric acid cleavage and thin layer chromatography. EPR-spectra of the aerobically prepared enzyme exhibit the so-called 'desulpho-inhibited'-signal, known from chemically modified forms of molybdenum containing proteins. Anaerobically prepared samples show both, the signals arising from the active molybdenum-cofactor as well as from the two [2Fe-2S]-clusters. According to metal-, cofactor-, and subunit-composition, the enzyme resembles the members of the xanthine oxidase family. Nevertheless, the melting point and long-term thermostability of the protein are outstanding and perfectly in tune with the growth temperature of S. acidocaldarius (80 degrees C). The findings suggest the enzyme to function as a glyceraldehyde oxidoreductase in the course of the nonphosphorylated Entner-Doudoroff pathway and thereby may attribute a new physiological role to this class of enzyme.  (+info)

(6/1596) Zyxin and vinculin distribution at the cell-extracellular matrix attachment complex (CMAX) in corneal epithelial tissue are actin dependent.

Avian embryonic corneal epithelia are two cell layers thick. If isolated without (-) basal lamina, the basal cells have unorganized actin and project cytoplasmic protrusions termed blebs. The actin-based cytoskeleton at the cell-extracellular matrix junction (termed the actin cortical mat) is disrupted. These epithelia respond to soluble extracellular matrix molecules by reorganizing the actin cortical mat. Sheets of epithelia were isolated + or -basal lamina. Epithelia isolated -basal lamina were cultured +/- laminin-1 and/or +/- cytochalasin D (CD). The intracellular localization of zyxin, vinculin, paxillin, focal adhesion kinase, and tensin was determined using indirect immunohistochemistry. Protein levels were determined by Western blot analysis. Zyxin and vinculin were concentrated in two areas of the tissue. The interface between the upper cell layer (periderm) and the basal cells. The second area of concentration was at the inferior 1-4 microns of the basal cells in an area with multiple actin bundles termed the actin cortical mat. The actin bundles align toward zyxin and vinculin that were located near basal lateral membranes. Zyxin was displaced from the basal compartment of blebbing basal cells. In contrast tensin, vinculin and focal adhesion kinase were found diffusely throughout the blebs. Zyxin and vinculin redistributed to the basal-lateral membranes as actin bundles reorganized in laminin-stimulated epithelia. In contrast to the altered protein distribution, extractable protein levels were similar in blebbing and laminin-stimulated epithelia. Zyxin, vinculin, and other associated proteins were disrupted in the CD-treated tissues and do not colocalize with each other or CD-induced actin aggregates. The intracellular localization of zyxin and vinculin were concentrated in distinct areas along the inferior basolateral membranes of basal cells termed the cell-extracellular matrix attachment complex (CMAX). The distribution of CMAX proteins was dependent upon actin bundle organization.  (+info)

(7/1596) Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties.

In our previous paper, we reported a mutant of recombinant Myrothecium verrucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [Shimizu, A. et al. (1999) Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.  (+info)

(8/1596) Degradation of hepatic zinc-thionein after parenteral zinc administration.

A low-molecular-weight protein, zinc-thionein, a metallothionein, was implicated as having a regulatory function in zinc metabolism. The half-life (t 1/2) of hepatic zinc-thionein was determined by pulse-labelling with either L-[35S] cystine and/or 65Zn. In two experiments with L-[35S]cystine, the t 1/2 of zinc-thionein was 18h and 19h. Most of the soluble 35S-labelled hepatic proteins had a t 1/2 of 4 days. The t 1/2 of zinc-thionein calculated by using 65Zn was 20h. The close similarity between the calculated and measured t 1/2 values for zinc-thionein suggests that release of Zn2+ from zinc-thionein probably occurs simultaneously with degradation of the protein moiety.  (+info)