Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein.
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.
Hassan-Abdallah
Alshaimaa
A
Department of Biochemistry and Molecular Biology, College of Medicine, Drexel University, Philadelphia, Pennsylvania 19102, USA.
Bruckner
Robert C
RC
Zhao
Guohua
G
Jorns
Marilyn Schuman
MS
D001051:Apoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050879:Sarcosine Oxidase; D013053:Spectrophotometry
D001051:Apoenzymes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C090003:8-(methylsulfonyl)FAD; C023406:1-deaza-FAD; C017123:8-chloro-FAD; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D003545:Cysteine