Structure of the Sodium Borohydride-Reduced N-(Cyclopropyl)glycine Adduct of the Flavoenzyme Monomeric Sarcosine Oxidase^,

Abstract

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.

Use of pH and Kinetic Isotope Effects To Establish Chemistry as Rate-Limiting in Oxidation of a Peptide Substrate by LSD1

“…LSD1 is inactivated by cyclopropyl substrates (25), and inactivation by such compounds is frequently taken as evidence for radical intermediates (34). However, a number of flavoprotein oxidases for which hydride transfer is the accepted mechanism can be inactivated by cyclopropyl substrates (35, 36). The kinetic parameter k cat /K 21-mer exhibits a pH optimum of 8.7 in the studies described here 3.…”

Oxidation of amines by flavoproteins

“…In the process a modified flavin is formed that is unstable, but it can be reduced by sodium borohydride to a form sufficiently stable for structure determination [58]. The electron density and mass of this modified flavin is consistent with formation of a five member ring involving the flavin N5 and C4a.…”

“…Since opening of cyclopropyl rings during a redox reaction is typically taken as evidence for a radical intermediate [59–61], the mechanism of Scheme 9 was proposed. The relevance of this reaction to the mechanism of oxidation of sarcosine during turnover was questioned at the time because the inactivation reaction was much slower than normal turnover [58]. In addition, both cholesterol oxidase and methanol oxidase are inactivated by cyclopropyl compounds [62, 63], but the evidence favors a hydride transfer mechanism for the glucose oxidase/cholesterol oxidase/methanol oxidase family of enzymes [64–66].…”

Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

“…24 Despite this similarity, quite different mechanisms have been proposed for the sarcosine oxidase family compared to DAAO, including radical, nucleophilic, and hydride transfer mechanisms. [25][26][27] We selected E. coli Nmethyltryptophan oxidase (MTOX) as a representative member of this family for study, and used the slow substrate sarcosine. The k cat and k cat /K m pH profiles for sarcosine are consistent with a requirement that the nitrogen of the substrate be unprotonated and a group in the free enzyme, probably the flavin N(3), be protonated for activity.…”