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 E. coli strain. A timedependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to about 80% of the level observed with native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from native MSOX. The reconstitution reaction exhibits apparent second order kinetics (k = 139 M −1 min −1 , 23 ºC) 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.Although flavins often function as noncovalently bound prosthetic groups, a growing number of flavoenzymes have been found to contain covalently bound flavin. This diverse group of enzymes perform a remarkable range of reactions, including ion pumping, antibiotic synthesis and metabolism of biogenic amines (1-3). Monomeric sarcosine oxidase (MSOX 1 ) is a prototypical member of a recently discovered family of amine-oxidizing enzymes that all contain covalently bound flavin (4-7). MSOX is an inducible bacterial enzyme that plays an important role in the catabolism of sarcosine (N-methylglycine), a common soil metabolite (8). The enzyme is widely used in the clinical evaluation of renal function (9). MSOX catalyzes the oxygen-dependent demethylation of sarcosine to yield glycine, formaldehyde and hydrogen peroxide. High resolution crystal structures are available for free MSOX and complexes of the enzyme with competitive inhibitors (10,11). MSOX contains 1 mol of FAD, attached to the protein via a thioether linkage between the 8α-methyl group of the isoalloxazine ring and Cys315 (8α-S-cysteinyl-FAD) (12) (see Table 1 for structure). The same covalent linkage is found for FAD in monoamine oxidase A and B, mammalian enzymes that exhibit 20% sequence identity with MSOX. In other enzymes, tyrosine or histidine may replace cysteine as *To whom requests for reprints should be addressed. Phone: (215) Covalent flavin atta...
We report the application of phosphoramidate pronucleotide (ProTide) technology to the antiviral agent carbocyclic L-d4A (L-Cd4A). The phenyl methyl alaninyl parent ProTide of L-Cd4A was prepared by Grignard-mediated phosphorochloridate reaction and resulted in a compound with significantly improved anti-HIV (2600-fold) and HBV activity. We describe modifications of the aryl, ester, and amino acid regions of the ProTide and how these changes affect antiviral activity and metabolic stability. Separate and distinct SARs were noted for HIV and HBV. Additionally, ProTides were prepared from the D-nucleoside D-Cd4A and the dideoxy analogues L-CddA and D-CddA. These compounds showed more modest potency improvements over the parent drug. In conclusion, the ProTide approach is highly successful when applied to L-Cd4A with potency improvements in vitro as high as 9000-fold against HIV. With a view to preclinical candidate selection we carried out metabolic stability studies using cynomolgus monkey liver and intestinal S9 fractions.
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