Absolute stereochemistry of flavins in enzyme-catalyzed reactions

Abstract

The 8-demethyl-8-hydroxy-5-deaza-5-carba analogues of FMN and FAD have been synthesized. Several apoproteins of flavoenzymes were successfully reconstituted with these analogues. This and further tests established that these analogues could serve as general probes for flavin stereospecificity in enzyme-catalyzed reactions. The method used by us involved stereoselective introduction of label on one enzyme combined with transfer to and analysis on a second enzyme. Using as a reference glutathione reductase from human erythrocytes for which the absolute stereochemistry of catalysis is known from X-ray studies [Pai, E. F., & Schulz, G. E. (1983) J. Biol. Chem. 258, 1752-1758], we were able to determine the absolute stereospecificities of other flavoenzymes. We found that glutathione reductase (NADPH), general acyl-CoA dehydrogenase (acyl-CoA), mercuric reductase (NADPH), thioredoxin reductase (NADPH), p-hydroxybenzoate hydroxylase (NADPH), melilotate hydroxylase (NADH), anthranilate hydroxylase (NADPH), and glucose oxidase (glucose) all use the re face of the flavin ring when interacting with the substrates given in parentheses.

Nucleotide Sequence of the FAD Synthetase Gene fromCorynebacterium ammoniagenesand Its Expression inEscherichia coli

“…3). The N-terminal 18 amino acid sequence deduced from the nucleotide sequence was identical with that of the purified FAD synthetase eMDIXYGTAAVPKDLXNXA 18) reported by Manstein et al 4) The corresponding gene product consisted of 338 amino acids and had a calculated molecular weight of 37,712. This value was also consistent with that (38 kDa) measured as the molecular mass of the FAD synthetase of B. ammoniagenes by SDS-PAGE.…”

“…This value was also consistent with that (38 kDa) measured as the molecular mass of the FAD synthetase of B. ammoniagenes by SDS-PAGE. 4 ) The overall G+C content within the ORF was 53.5%, which was about same as that (53.7-54.6%) for the total genomic DNA of C. ammoniagenes. 5 ) The G + C contents at the I st, 2nd, and 3rd codon positions of the ORF were 64.0, 41.3, and 55.2%, respectively.…”

4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3

“…From stereochemical studies it is known that the nicotinamide part of the reduced pyridine nucleotide cofactor binds at the re-side of the flavin ring allowing rapid hydride transfer (Manstein et al, 1986). Chemical modification studies (van Berkel and Miiller, 1991) and model building (van Berkel et al, 1988) have indicated that the pyrophosphate moiety of the pyridine nucleotide cofactor most probably binds in a cleft between the FAD-binding domain and the substrate-binding domain.…”

The Puzzle of Ligand Binding to Corynebacterium ammoniagenes FAD Synthetase

“…In the adenylylation process, FMN is proposed to bind after ATP and the PP i to be released preceding FAD (10). The two enzymatic activities differ in their specificity for divalent cations, optimal pH, and temperature (8,11,12). The presence of Mg 2ϩ improves the turnover of both processes but, although low concentrations (Ͻ1 mM) enhance the kinase activity, much larger concentrations (ϳ10 mM) are required for maximal FAD production (8).…”

“…1A). Eukaryotes generally use two different enzymes for FMN and FAD production (1-7), whereas most prokaryotes depend on a single bifunctional enzyme, the FAD synthetase (FADS) (8,9).…”