Covalent flavinylation of monomeric sarcosine oxidase: identification of a residue essential for holoenzyme biosynthesis

Biochemistry
Alshaimaa Hassan-AbdallahM S Jorns

Abstract

FAD in monomeric sarcosine oxidase (MSOX) is covalently linked to the protein by a thioether linkage between its 8alpha-methyl group and Cys315. Covalent flavinylation of apoMSOX has been shown to proceed via an autocatalytic reaction that requires only FAD and is blocked by a mutation of Cys315. His45 and Arg49 are located just above the si-face of the flavin ring, near the site of covalent attachment. His45Ala and His45Asn mutants contain covalently bound FAD and exhibit catalytic properties similar to wild-type MSOX. The results rule out a significant role for His45 in covalent flavinylation or sarcosine oxidation. In contrast, Arg49Ala and Arg49Gln mutants are isolated as catalytically inactive apoproteins. ApoArg49Ala forms a stable noncovalent complex with reduced 5-deazaFAD that exhibits properties similar to those observed for the corresponding complex with apoCys315Ala. The results show that elimination of a basic residue at position 49 blocks covalent flavinylation but does not prevent noncovalent flavin binding. The Arg49Lys mutant contains covalently bound FAD, but its flavin content is approximately 4-fold lower than wild-type MSOX. However, most of the apoprotein in the Arg49Lys preparation is reconstitutable with...Continue Reading

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Citations

Jul 26, 2014·Journal of Chemical Theory and Computation·Anthony Bucci, Cameron F Abrams
Jun 23, 2016·Journal of the American Chemical Society·Isita JhulkiTadhg P Begley
Feb 2, 2019·Mikrochimica Acta·Tailin LiuKang Li
Apr 5, 2021·Archives of Biochemistry and Biophysics·Majd LahhamSilvia Wallner

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