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Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein
Journal article   Open access   Peer reviewed

Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein

Alshaimaa Hassan-Abdallah, Robert C Bruckner, Guohua Zhao and Marilyn Schuman Jorns
Biochemistry (Easton), v 44(17), pp 6452-6462
03 May 2005
DOI: https://doi.org/10.1021/bi047271x
PMID: 15850379
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1021/bi047271xView
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Abstract

Oxidoreductases, N-Demethylating - genetics Oxidoreductases, N-Demethylating - isolation & purification Apoenzymes - biosynthesis Recombinant Proteins - biosynthesis Apoenzymes - isolation & purification Recombinant Proteins - isolation & purification Flavin-Adenine Dinucleotide - metabolism Sarcosine Oxidase Apoenzymes - metabolism Cysteine - metabolism Escherichia coli - growth & development Oxidoreductases, N-Demethylating - metabolism Binding Sites Recombinant Proteins - metabolism Escherichia coli - enzymology Flavin-Adenine Dinucleotide - isolation & purification Flavin-Adenine Dinucleotide - analogs & derivatives Bacillus - genetics Recombinant Proteins - genetics Flavin-Adenine Dinucleotide - chemical synthesis Mutagenesis Bacillus - enzymology Escherichia coli - genetics Apoenzymes - genetics Kinetics Oxidoreductases, N-Demethylating - biosynthesis Spectrophotometry
The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.

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Web of Science research areas
Biochemistry & Molecular Biology
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