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

Schräder, Thomas; Andreesen, Jan R.

doi:10.1111/j.1432-1033.1993.tb18428.x

Cited by 31 publications

(31 citation statements)

References 39 publications

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“…The spectrum of F1 exhibits two strong bands in the visible region, centered at 365 nm and 455 nm, which are characteristic of enzyme-bound FAD in the oxidized state. The value of 6.5 for the A 280 /A 455 ratio is consistent with values reported in the literature (28). The spectrum of F2 is significantly altered compared with that of F1.…”

Section: Single-site Oxidation Of Cys108 Distinguishes F2 From F1supporting

confidence: 81%

Single-Site Oxidation, Cysteine 108 to Cysteine Sulfinic Acid, ind-Amino Acid Oxidase fromTrigonopsis variabilisand Its Structural and Functional Consequences

Slavica

Dib

Nidetzky

2005

Appl Environ Microbiol

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One of the primary sources of enzyme instability is protein oxidative modification triggering activity loss or denaturation. We show here that the side chain of Cys108 is the main site undergoing stress-induced oxidation in Trigonopsis variabilis D-amino acid oxidase, a flavoenzyme employed industrially for the conversion of cephalosporin C. High-resolution anion-exchange chromatography was used to separate the reduced and oxidized protein forms, which constitute, in a molar ratio of about 3:1, the active biocatalyst isolated from the yeast. Comparative analysis of their tryptic peptides by electrospray tandem mass spectrometry allowed unequivocal assignment of the modification as the oxidation of Cys108 into cysteine sulfinic acid. Cys108 is likely located on a surface-exposed protein region within the flavin adenine dinucleotide (FAD) binding domain, but remote from the active center. Its oxidized side chain was remarkably stable in solution, thus enabling the relative biochemical characterization of native and modified enzyme forms. The oxidation of Cys108 causes a global conformational response that affects the protein environment of the FAD cofactor. In comparison with the native enzyme, it results in a fourfold-decreased specific activity, reflecting a catalytic efficiency for reduction of dioxygen lowered by about the same factor, and a markedly decreased propensity to aggregate under conditions of thermal denaturation. These results open up unprecedented routes for stabilization of the oxidase and underscore the possible significance of protein chemical heterogeneity for biocatalyst function and stability.

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Section: Single-site Oxidation Of Cys108 Distinguishes F2 From F1supporting

confidence: 81%

Single-Site Oxidation, Cysteine 108 to Cysteine Sulfinic Acid, ind-Amino Acid Oxidase fromTrigonopsis variabilisand Its Structural and Functional Consequences

Slavica

Dib

Nidetzky

2005

Appl Environ Microbiol

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“…In contrast, thiol-modifying reagents, which can strongly inhibit many DAOs, did not significantly inhibit RxDAO. Such reagents inactivate TvDAO and RgDAO by the oxidation of C298 and C208, respectively (36,37). However, no cysteine residue is conserved at the corresponding positions of RxDAO or is found near the active site.…”

Section: Discussionmentioning

confidence: 99%

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

Takahashi

Furukawara

Omae

et al. 2014

Appl Environ Microbiol

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D-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic D-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested D-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain D-amino acids, with the highest specific activity toward D-valine and catalytic efficiency (k cat /K m ) toward D-leucine. Substrate inhibition was observed in the case of D-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T 50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain D-amino acids, and as a scaffold to generate a novel DAO via protein engineering.T he flavoenzyme D-amino acid oxidase (DAO; EC 1.4.3.3) catalyzes the oxidative deamination of neutral and basic D-amino acids but not of acidic D-amino acids, which are substrates of D-aspartate oxidase (DDO). DAO was first identified in pig kidney (pkDAO) by Krebs in 1935 (1) and subsequently has been found mainly in eukaryotic organisms, ranging from fungi to humans (2). These eukaryotic DAOs have been extensively studied as a model of the oxidase-dehydrogenase class of flavoproteins. In fungi, the enzyme plays a role in the assimilation of D-amino acids for cell growth and also in the detoxification of D-amino acid toxicity (2, 3). In vertebrates, this enzyme is mainly localized in the liver and kidney, and it metabolizes both exogenous and endogenous D-amino acids (2). In addition, the enzyme plays a specific role in the regulation of D-serine, a coagonist for the N-methyl-Daspartate receptor, in the brain (2).In contrast, for a long time, it was thought that DAO did not exist in prokaryotic organisms. However, this enzyme was recently identified in Arthrobacter protophormiae (ApDAO) and Streptomyces coelicolor (ScDAO) (4, 5). In addition to these bacterial species, a search of a prokaryotic genome database revealed a wide distribution of DAO homologous proteins in bacteria, especially a...

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“…A paper reported also on reactive histidine(s) in T6DAAO: in this case, too, a possible involvement of the residue in catalysis was envisaged [62]. As second putative essential residue, cysteine reactivity was examined in T6DAAO [63] and in RgDAAO [64]. For the latter, a single reactive cysteine was identified, clearly involved in FAD binding.…”

Section: Site-directed Mutagenesis Studiesmentioning

confidence: 99%

D-Amino acid oxidase: new findings

Pilone¹

2000

CMLS, Cell. Mol. Life Sci.

187

177

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The most recent research on D-amino acid oxidases and D-amino acid metabolism has revealed new, intriguing properties of the flavoenzyme and enlighted novel biotechnological uses of this catalyst. Concerning the in vivo function of the enzyme, new findings on the physiological role of D-amino acid oxidase point to a detoxifying function of the enzyme in metabolizing exogenous D-amino acids in animals. A novel role in modulating the level of D-serine in brain has also been proposed for the enzyme. At the molecular level, site-directed mutagenesis studies on the pig kidney D-amino acid oxidase and, more recently, on the enzyme from the yeast Rhodotorula gracilis indicated that the few conserved residues of the active site do not play a role in acid-base catalysis but rather are involved in substrate interactions. The three-dimensional structure of the enzyme was recently determined from two different sources: at 2.5-3.0 A resolution for DAAO from pig kidney and at 1.2-1.8 A resolution for R. gracilis. The active site can be clearly depicted: the striking absence of essential residues acting in acid-base catalysis and the mode of substrate orientation into the active site, taken together with the results of free-energy correlation studies, clearly support a hydrid transfer type of mechanism in which the orbital steering between the substrate and the isoalloxazine atoms plays a crucial role during catalysis.

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

Cited by 31 publications

References 39 publications

Single-Site Oxidation, Cysteine 108 to Cysteine Sulfinic Acid, ind-Amino Acid Oxidase fromTrigonopsis variabilisand Its Structural and Functional Consequences

Single-Site Oxidation, Cysteine 108 to Cysteine Sulfinic Acid, ind-Amino Acid Oxidase fromTrigonopsis variabilisand Its Structural and Functional Consequences

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

D-Amino acid oxidase: new findings

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