scholarly journals Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP+ oxidoreductase activity toward NADPH

2015 ◽  
Vol 125 (1-2) ◽  
pp. 321-328 ◽  
Author(s):  
Daisuke Seo ◽  
Hiroshi Naito ◽  
Erika Nishimura ◽  
Takeshi Sakurai
Keyword(s):  
Bacillus Subtilis ◽  
Flavin Adenine Dinucleotide ◽  
Prosthetic Group ◽  
Oxidoreductase Activity

scholarly journals Heme A Synthase Enzyme Functions Dissected by Mutagenesis of Bacillus subtilis CtaA

2005 ◽  
Vol 187 (24) ◽  
pp. 8361-8369 ◽  
Author(s):  
Lars Hederstedt ◽  
Anna Lewin ◽  
Mimmi Throne-Holst
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Mechanism ◽  
Enzyme Reaction ◽  
Prosthetic Group ◽  
Histidine Residues ◽  
Functional Roles ◽  
Heme A Synthase ◽  
Respiratory Oxidases

ABSTRACT Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.

8.alpha.-(O-Tyrosyl)flavin adenine dinucleotide, the prosthetic group of bacterial p-cresol methylhydroxylase

Biochemistry ◽  
1981 ◽  
Vol 20 (11) ◽  
pp. 3068-3075 ◽  
Author(s):  
William McIntire ◽  
Dale E. Edmondson ◽  
David J. Hopper ◽  
Thomas P. Singer
Keyword(s):  
Flavin Adenine Dinucleotide ◽  
Prosthetic Group

scholarly journals MORPHOLOGICAL AND CYTOCHEMICAL IDENTIFICATION OF THE GOLGI APPARATUS

1956 ◽  
Vol 2 (3) ◽  
pp. 263-280 ◽  
Author(s):  
C. H. U. Chu ◽  
C. A. Swinyard
Keyword(s):  
Golgi Apparatus ◽  
Xanthine Oxidase ◽  
Aldehyde Dehydrogenase ◽  
Flavin Adenine Dinucleotide ◽  
Physiological Condition ◽  
Specific Protein ◽  
Living Cells ◽  
Silver Impregnation ◽  
Prosthetic Group ◽  
Golgi Network

1. A modification of Elftman's direct silver method reveals both the lipochondria of Baker and the network of Golgi in the same cell. For purpose of distinction, it is proposed to call Baker's lipochondria the nucleopetal fraction, the Golgi network the nucleofugal fraction of the Golgi apparatus. 2. The nucleopetal fraction is located closer to the nucleus. It is spherical in shape and appears black in color. The nucleofugal fraction is located farther away from the nucleus. It is reticular in form and appears brown in color after silver impregnation by the modified Elftman's method. 3. These two fractions are separate entities. The network of Golgi is not due to deposition of silver on lipochondria. Lipochondria do not represent Golgi apparatus in living cells. 4. Aldehydes facilitate the demonstration of the nucleofugal fraction. Based on the circumstantial evidence presented, it appears that aldehyde dehydrogenase composed of a specific protein bound to a prosthetic group of flavin-adenine dinucleotide may be concentrated in this fraction. 5. Aldehyde dehydrogenase also functions as xanthine oxidase. It is suggested as a working hypothesis that under physiological condition, one of the functions of the nucleofugal fraction (Golgi network) is concerned with purine metabolism of nucleoproteins.

scholarly journals The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

2007 ◽  
Vol 190 (5) ◽  
pp. 1546-1553 ◽  
Author(s):  
Simon Grill ◽  
Simone Busenbender ◽  
Matthias Pfeiffer ◽  
Uwe Köhler ◽  
Matthias Mack
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Sus Scrofa ◽  
Flavin Adenine Dinucleotide ◽  
Functional Expression ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Sensitive Bacterium ◽  
Antibiotic Effect

ABSTRACT Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5′-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa d-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, k cat/Km = 1.7 10−2 μM−1 s−1; riboflavin, k cat/Km = 7.5 10−3 μM−1 s−1). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, k cat/Km = 1.3 10−2 μM−1 s−1; riboflavin, k cat/Km = 1.3 10−2 μM−1 s−1). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.

scholarly journals Purification, Characterization, and Mechanism of a Flavin Mononucleotide-Dependent 2-Nitropropane Dioxygenase fromNeurospora crassa

1998 ◽  
Vol 64 (3) ◽  
pp. 1029-1033 ◽  
Author(s):  
Natalia Gorlatova ◽  
Marek Tchorzewski ◽  
Tatsuo Kurihara ◽  
Kenji Soda ◽  
Nobuyoshi Esaki
Keyword(s):  
Molecular Weight ◽  
Aromatic Compounds ◽  
Carbonyl Compounds ◽  
Superoxide Anion ◽  
Flavin Adenine Dinucleotide ◽  
Enzyme Reaction ◽  
Flavin Mononucleotide ◽  
Prosthetic Group ◽  
Reaction Stoichiometry ◽  
Better Than

ABSTRACT A nitroalkane-oxidizing enzyme was purified to homogeneity fromNeurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an18O2 atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH3CH(NO2)-CH3 + O2→2CH3COCH3 + 2HNO2, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of theNeurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenulaenzyme. Both of these enzymes are flavoenzymes; however, theNeurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.

scholarly journals Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134

2007 ◽  
Vol 190 (5) ◽  
pp. 1615-1619 ◽  
Author(s):  
Sara Mae Belchik ◽  
Luying Xun
Keyword(s):  
Flavin Adenine Dinucleotide ◽  
Chemical Reduction ◽  
Quinone Reductase ◽  
Cupriavidus Necator ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Oxidoreductase Activity ◽  
Quinone Reduction ◽  
His Tag ◽  
Toxic Pollutant

ABSTRACT The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH2, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (tcpX and tcpB) were cloned, overexpressed, and purified in Escherichia coli. TcpX was purified as a C-terminal His tag fusion (TcpXH) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpXH had no activity toward NADPH or riboflavin. Coupling of TcpXH and TcpA demonstrated that TcpXH provided FADH2 for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpXH in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.

scholarly journals Dihydropteridine Reductase as an Alternative to Dihydrofolate Reductase for Synthesis of Tetrahydrofolate in Thermus thermophilus

2004 ◽  
Vol 186 (2) ◽  
pp. 351-355 ◽  
Author(s):  
Valérie Wilquet ◽  
Mark Van de Casteele ◽  
Daniel Gigot ◽  
Christianne Legrain ◽  
Nicolas Glansdorff
Keyword(s):  
Dihydrofolate Reductase ◽  
Reductase Activity ◽  
Thermus Thermophilus ◽  
Personal Communication ◽  
Dihydropteridine Reductase ◽  
Active Site Residues ◽  
Prosthetic Group ◽  
Oxidoreductase Activity ◽  
Gene Coding ◽  
Genome Nucleotide Sequence

ABSTRACT A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH Tt ] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH Tt appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.

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