Covalent flavinylation of monomeric sarcosine oxidase: identification of a residue essential for holoenzyme biosynthesis.
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 FAD in a reaction that exhibits kinetic parameters similar to those observed for flavinylation of wild-type apoMSOX. Although covalent flavinylation is scarcely affected, the specific activity of the Arg49Lys mutant is only 4% of that observed with wild-type MSOX. The results show that a basic residue at position 49 is essential for covalent flavinylation of MSOX and suggest that Arg49 also plays an important role in sarcosine oxidation.
Hassan-Abdallah
Alshaimaa
A
Department of Biochemistry and Molecular Biology, Drexel University, College of Medicine, Philadelphia, Pennsylvania 19102, USA.
Zhao
Guohua
G
Jorns
Marilyn Schuman
MS
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D020035:Holoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D050879:Sarcosine Oxidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D005415:Flavins; D020035:Holoenzymes; D050879:Sarcosine Oxidase