Structure of the sodium borohydride-reduced N-(cyclopropyl)glycine adduct of the flavoenzyme monomeric sarcosine oxidase.
Monomeric sarcosine oxidase (MSOX) is a flavoprotein that contains covalently bound FAD [8a-(S-cysteinyl)FAD] and catalyzes the oxidation of sarcosine (N-methylglycine) and other secondary amino acids, such as l-proline. Our previous studies showed that N-(cyclopropyl)glycine (CPG) acts as a mechanism-based inactivator of MSOX [Zhao, G., et al. (2000) Biochemistry 39, 14341-14347]. The reaction results in the formation of a modified reduced flavin that can be further reduced and stabilized by treatment with sodium borohydride. The borohydride-reduced CPG-modified enzyme exhibits a mass increase of 63 +/- 2 Da as compared with native MSOX. The crystal structure of the modified enzyme, solved at 1.85 A resolution, shows that FAD is the only site of modification. The modified FAD contains a fused five-membered ring, linking the C(4a) and N(5) atoms of the flavin ring, with an additional oxygen atom bound to the carbon atom attached to N(5) and a tetrahedral carbon atom at flavin C(4) with a hydroxyl group attached to C(4). On the basis of the crystal structure of the borohydride-stabilized adduct, we conclude that the labile CPG-modified flavin is a 4a,5-dihydroflavin derivative with a substituent derived from the cleavage of the cyclopropyl ring in CPG. The results are consistent with CPG-mediated inactivation in a reaction initiated by single electron transfer from the amine function in CPG to FAD in MSOX, followed by collapse of the radical pair to yield a covalently modified 4a,5-dihydroflavin.
Chen
Zhi-wei
ZW
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Zhao
Gouhua
G
Martinovic
Suzana
S
Jorns
Marilyn Schuman
MS
Mathews
F Scott
FS
D001894:Borohydrides / Q000737:chemistry; D018360:Crystallography, X-Ray; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010456:Peptides, Cyclic / Q000737:chemistry*; D050879:Sarcosine Oxidase / Q000737:chemistry*
D001894:Borohydrides; D010456:Peptides, Cyclic; D005182:Flavin-Adenine Dinucleotide; C025364:sodium borohydride; C062238:cyclo(prolylglycyl); D050879:Sarcosine Oxidase; D005998:Glycine