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![[Figure 4]](ag5031fig4_combined_mag.jpg)
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Figure 4
Spectra, synthesis, reaction and structure of 5-deaza-FMN. (a) UV–Vis spectra of FMN in Hmo and its Y128F mutant: (i) FMNox in Hmo (370/450 nm, green line), (ii) acyl-FMNred in the Y128F mutant [340 nm, blue line; addition of phenylpyruvate (PPY) for 2 h], (iii) acylated FMN in redissolved Y128F mutant crystals/crystalloids soaked with PPY (acyl-FMNred, 340 nm, dotted blue line), (iv) FMNred in Hmo [a shoulder at 330 nm, red; addition of phenyllactate (PLA)]. (b) (i) Chemical synthesis and LC purification of 5-deazariboflavin (the inset shows the mass spectrum of 5-deazariboflavin), (ii) enzymatic synthesis and LC purification of 5-deaza-FMN (the inset shows the mass spectrum of 5-deaza-FMN). (c) Enzymatic reactions of Hmo and its Y128F mutant harboring 5-deaza-FMN: (i) enzymatic reaction with Hmo harboring 5-deaza-FMN in the presence of S-mandelate, (ii) enzymatic reaction with the Y128F mutant harboring 5-deaza-FMN in the presence of S-mandelate, (iii) control reaction of wild-type Hmo in the presence of S-mandelate, (iv) control reaction of the Y128F mutant in the presence of S-mandelate. (d, e) Crystal structures of the Y128F mutant harboring 5-deaza-FMN soaked with S-mandelate (d) or phenyllactate (e), which have been transformed into benzoylformate or phenylpyruvate, respectively. Unlike FMN in the wild type or the Y128F mutant, no electron density emerges at the top of C5 or between C′α and C5. (f) The structure of an α-mandelamide–N5-FMNred adduct in the crystal of the R163L mutant soaked with nondecarboxylable α-mandelamide (the chemical structure is shown). The flavin adduct is on the si-face of the isoalloxazine ring. The 2Fo − Fc electron-density map is contoured at 2σ. The unbiased Fo − Fc difference electron-density map is contoured at 4σ in positive electron density. Free ligands, FMN, FMN adducts and active-site residues are colored cyan, yellow, orange and green, respectively. See Supplementary Figs. S2(j)–S2(m) for stereoviews and 2Fo − Fc difference electron-density maps. |
![[Figure 4]](ag5031fig4_combined.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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