Biosynthesis of Covalently Bound Flavin: Isolation and in Vitro Flavinylation of the Monomeric Sarcosine Oxidase Apoprotein

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Abstract
The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8α-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 ∼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 °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 8α-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 (8α-S-cysteinyl), a difference that may account for its ∼10-fold lower catalytic activity.

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