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. 2021 May 6;10:e59295. doi: 10.7554/eLife.59295

Figure 5. Recombinant Rha1 and Rha2 metabolize 4-HNE to 4-HNA.

Figure 5.

(A) Phyre2 structural homology models predict that asparagine-47 interacts with the FMN in Rha1 (left) and tyrosine-195 coordinates NADPH in Rha2 (right). (B) Rates of NADPH oxidation (200 µM) by WT (blue) and mutant (orange) variants of Rha1 and Rha2 in the presence of 4-HNE. (C) Rate of NADPH oxidation (200 µM) by human enzyme AKR1C1 in the presence of 4-HNE. (D) NADPH oxidation rate by Rha1 and Rha2 in the presence of 640 µM final concentration of various aldehydes. (E) TLC plates showing the migration of reaction contents of Lane 1: 4-HNE, Lane 2: 1,4-DHN, and Lanes 3–6: indicated enzymes with 4-HNE in the absence and presence of NADPH after 1 hr of reaction at room temperature. 4-HNE -- black arrow, 4-HNA -- red arrow, 1,4-DHN -- blue arrow. (F) Diagram of 4-HNE to 4-HNA conversion. Data in (B), (C), (D) and (E) are representative of at least three independent experiments.