Extended Data Fig. 6: SznF is an iron-dependent monooxygenase. | Nature

Extended Data Fig. 6: SznF is an iron-dependent monooxygenase.

From: An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin

Extended Data Fig. 6

a, When 1 mM l-arginine, 80 μM SznF, 20 μM PMS and 5 mM NADH were incubated at room temperature for 1 h, only trace amounts of a mass corresponding to l-hydroxyarginine (EIC ([M − H]) = 189.0993) were observed. No masses corresponding to l-hydroxycitrulline (EIC ([M − H]) = 190.0883), l-dihydroxyarginine (EIC ([M − H]) = 205.0942) or the l-nitrosocitrulline (EIC([M − H]) = 203.0786) were observed. b, The [M − H] mass spectrum of 3 generated when [15N413C6]l-NMA and unlabelled l-NMA were mixed in the same SznF reaction mixture. c, Testing the metal dependence of SznF. 80 μM of apo-SznF was incubated with 200 μM of various divalent metals, 20 μM PMS, 1 mM l-NMA and 5 mM NADH for 1 h at room temperature. The EIC traces were generated with a 5 p.p.m. window. d, Oxygen was rapidly consumed in the presence of l-NMA and SznF as measured by an optode. SznF(E281A), which lacks a key predicted iron-binding residue in the central domain, failed to consume oxygen above background. The background consumption of oxygen arises from the non-enzymatic reduction of PMS by NADH. e, Incubating 18O2, 1 mM l-HMA (1) and 80 μM SznF at room temperature for 1 h resulted in labelling of two of the N-nitrosourea oxygens. MS/MS analysis revealed retention of the Nδ-OH (data not shown). f, Addition of H218O to an SznF assay mixture did not label the N-nitrosourea group. The expected [M − H] masses for Fmoc-3, [18O]Fmoc-3, [18O2]Fmoc-3 and [18O3]Fmoc-3 are 455.1572, 457.1615, 459.1657 and 461.1700, respectively. g, Addition of catalase or superoxide dismutase to the assay mixtures did not affect SznF-catalysed N-oxygenation as measured by the Griess assay. Data are mean ± s.d. of three replicates.

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