Improved purification, amino acid analysis and molecular weight of homogeneous d-amino acid oxidase from pig kidney

Curti, Bruno; Ronchi, Severino; Branzoli, Umberto; Ferri, Giuseppina; Williams, Charles H.

doi:10.1016/0005-2744(73)90409-9

Cited by 88 publications

(38 citation statements)

References 20 publications

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“…It was discussed that these analogs may be good steric replacements for riboflavin but not catalytic substitutes (42). Our experiments with RoFAD-reconstituted porcine DAAO apoenzyme (10) showed that RoFAD indeed is not functional. In the case of RoFAD-DAAO, we speculate that it is not the altered redox potential of RoFAD that is primarily responsible for inactive DAAO but rather the fact that RoFAD is not properly positioned in the enzyme.…”

Section: Discussionmentioning

confidence: 77%

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

et al. 2008

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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. ). 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.Riboflavin (vitamin B 2 ) analogs have the potential to serve as basic structures for the development of novel anti-infectives (3). Consequently, both the mode of action of riboflavin analogs and the mechanism of resistance to these compounds are of substantial interest. Only very few natural riboflavin analogs are known (24). Moreover, the only known organism to produce a riboflavin analog with antibiotic activity is the grampositive bacterium Streptomyces davawensis (32).S. davawensis synthesizes the anti-vitamin 8-dimethyl-amino-8-demethyl-D-riboflavin, or roseoflavin ( Fig. 1), from riboflavin (27) via 8-amino-and 8-methylamino-8-demethyl-D-riboflavin (22). Roseoflavin is toxic to gram-positive but also to gramnegative bacteria if the compound is able to enter the cell (16). The molecular basis for roseoflavin toxicity is not clear. In all organisms, riboflavin serves as the direct precursor for the cofactors (ribo)flavin-5Ј-monophosphate (FMN) and flavin adenine dinucleotide (FAD) (12). FAD and (to a lesser extent) FMN are active components of flavoproteins, being involved in a wide range of redox and other reactions (15). FMN is synthesized from riboflavin by flavokinase (EC 2.7.1.26), and FAD is produced from FMN by FAD synthetase (EC 2.7.7.2) (2). In microorganisms, both enzymatic activities seem to be present in sufficient amounts, since only traces of free riboflavin are detectable in the cytoplasm (11). Accordingly, cytoplasmic roseoflavin may quickly be converted to the corresponding FMN/ FAD analogs roseoflavin-5Ј-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD). The synthesis of these inactive cofactors may explain the antibiotic activity of roseoflavin ( Fig. 1) (31, 36).S. davawensis is roseoflavin resistant, and the mechanism of self-resistance could involve a flavokinase/FAD synthetase with a high substrate specificity for riboflavin. Such an enzyme would not accept roseoflavin as...

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Section: Discussionmentioning

confidence: 77%

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

et al. 2008

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“…The basic properties of the purified enzyme were in good agreement with the data reported for D-amino acid oxidase from different sources [6-8, 10, 11, 391. A content of D-aminO acid oxidase of 2.7 2 0.3% of total soluble protein is the highest ever determined for this enzyme, followed by the yeast R. gracilis where it constitutes 0.3% of the cell protein [6,11,401. 7: variabilis represents the second source for D-amino acid oxidase from a microorganism yielding a homogeneous, stable, and highly active enzyme.…”

Section: Discussionmentioning

confidence: 89%

Evidence for the functional importance of Cys298 in D‐amino acid oxidase from Trigonopsis variabilis

Schräder

Andreesen

1993

European Journal of Biochemistry

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D-Amino acid oxidase from Trigonopsis variabilis was purified to homogeneity by a combination of freezekhawing, isoelectric precipitation and chromatography on Mono Q. This purification procedure required very little working effort. The homogeneous enzyme exhibited a ratio AZXJA45U of about 6.5 and was obtained in high yield (63%) and a good stability. Using D-methionine as a substrate, a specific activity of 120 U/mg was determined colorimetrically at 26 "C, corresponding to 185 U/mg polarographically at 37°C. Polyclonal antibodies were raised against the homogeneous protein and Western immunoblot analysis showed that the 39-kDa subunit can undergo defined cleavages at the carboxy terminus of amino acid positions 104, 106 and 108, leading to 27-kDa and 12-kDa fragments as revealed by SDSPAGE, which are still enzymically active in their native form. The enzyme was inactivated by all sulfhydryl-modifying reagents tested. Inactivation by 5,5'-dithiobis(-2-nitrobenzoate) was correlated with a modification of up to 2 mollmol protein of the six cysteine residues present in the monomer. Identification of the most reactive cysteine was achieved by inactivation of the enzyme with the fluorescent, sulfhydryl-modifying reagent monobromobimane. In the presence of a substrate amino acid, under anaerobic conditions, the protein could be protected from modification and, thus, inactivation by this reagent. Peptide mapping by reversephase chromatography of endoproteinase Glu-C-digested monobromobimane-labeled enzyme revealed one major fluorescence peak which was not obtained when the protein was modified in the presence of a substrate amino acid under anaerobic conditions. Isolation and sequencing of the labeled peptide led to the identification of Cys298 as the reactive cysteine residue. D-Amino acid oxidase catalyzes the oxidation of D-amino acids to their corresponding 2-imino acids which are subsequently hydrolysed non-enzymically to 2-0x0 acids and ammonia. Reoxidation of the enzyme by molecular oxygen is accompanied by the release of H,O,. The enzyme contains one FADlsubunit and belongs to the class of dehydrogenases/ oxidases [l]. This protein is of special importance in research and biotechnology 12-51 and was, therefore, subject to a diversity of investigations (for review see [2]). For a long time, the enzyme from pig kidney has been the only source of homogeneous D-amino acid oxidase 161. Recently, the enzyme from the yeast Rhodotorulu gracilis was purified to homogeneity and its properties were determined [7 -101. D-Amino acid oxidase activity was also detected in the yeast Trigonopsis vuriubilis, and the enzyme was purified to near homogeneity, but in contrast to R. gracilis the enzyme preparation showed a very low stability [ll]. Although the amino acid sequence of the enzyme is published [12] Chemical modification of D-amino acid oxidase from pig kidney by amino-acid-specific reagents led to the identification of histidine [15, 161, arginine [17], tyrosine [16, 181, lysine [18], methionine [19] and cysteine [20...

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“…Control experiments were performed using three enzymes that do not carry glycans. These were (heterologously expressed) human medium chain acyl CoA dehydrogenase (hMCAD) [29], mammalian DAAO [35] and yeast DAAO [36]. Figure 7(b) shows that all three proteins were retained to various extents in cell lysates but did not undergo fragmentation, suggesting that intracellular proteolysis is a specific feature of LAAO.…”

Section: Interaction Of Laao and Desialylated Laao With Jurkat Cellsmentioning

confidence: 99%

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

et al. 2006

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L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H 2 O 2 produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H 2 O 2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H 2 O 2 production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H 2 O 2 and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface.S. R. Ande, P. R. Kommoju contributed equally to this work.

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Improved purification, amino acid analysis and molecular weight of homogeneous d-amino acid oxidase from pig kidney

Cited by 88 publications

References 20 publications

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

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

Evidence for the functional importance of Cys298 in D‐amino acid oxidase from Trigonopsis variabilis

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

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