Fig. 4: FAD-dependent rearrangement of Z3AL to E2AL by lepidopteran Hi.
From: Convergent evolution of hexenal isomerases in Lepidoptera and plants
a–e, Increased conversion rate of E2AL with FAD addition. Purified recombinant Hi proteins—buffer (a), MsHi-1 (b), CvHi-1 (c), DpHi-1 (d) and BmHi-1 (e)—were incubated with Z3AL (0.2 mM) with or without FAD (1 mM). The proportion of E2AL emitted from total aldehydes (Z3AL + E2AL) was calculated. Statistically significant differences between treatments were assessed using ratio paired two-tailed t-tests: a: ηp² = 0.17, n = 6 biologically independent samples, t = 1.01, P = 0.36; b: ηp² = 0.72, n = 6, t = 3.55, P = 0.017; c: ηp² = 0.93, n = 5, t = 7.16, P = 0.002; d: ηp² = 0.90, n = 5, t = 5.94, P = 0.004; e: ηp² = 0.97, n = 3, t = 8.66, P = 0.013. The error bars are presented as mean values ± s.d. a′–e′, Representative extracted ion chromatograms (ion 69) from SPME–GC–MS for buffer (a′), MsHi-1 (b′), CvHi-1 (c′), DpHi-1 (d′) and BmHi-1 (e′). f,g, Superposition of the MsHi-1 structure with FAD docking (predicted using AlphaFold 3) with structures of aryl-alcohol oxidase from P. eryngii (PDB: 3FIM) (f) and glucose dehydrogenase from A. flavus (PDB: 4YNT) (g). f′,g′, Detailed view of the FAD-binding pocket shows an N-terminal residue that binds FAD and two conserved C-terminal catalytic residues of GMC oxidoreductases. These N-terminal residues H135 (MsHI-1), Y92 (3FIM) and G94 (4YNT) form a hydrogen bond (green dashed line) with the O4 atom of the FAD isoalloxazine ring. f′ and g′ show structural superimposition of the FAD-binding domain of MsHi-1 with homologous structures: 3FIM (f′) and 4YNT (g′). Structural alignment and r.m.s.d. calculations were performed using Mol* (https://molstar.org/). h, Sequence logos representing the conservation of catalytic residues among lepidopteran Hi homologues, and the positions correspond to MsHi-1. Mutated residues are highlighted in blue. Sequence logos were generated using WebLogo 3. i, Proportion of E2AL emitted from total aldehydes after incubation of Z3AL with either wild-type (WT) or mutant recombinant MsHi-1 (0.1 µg or 0.5 µg, as indicated by 5×). Significant differences between the buffer control, WT and mutant proteins were determined using one-way ANOVA (η² = 0.98, F7,25 = 144.3, P < 0.0001), followed by Tukey’s honestly significant difference post hoc test. Different letters indicate significant differences (P < 0.05). n = 6 biologically independent samples for buffer control; n = 5 for WT and H521A; n = 3 for H135A and H559A. The error bars are presented as mean ± s.d. j, Kinetic parameters of wild-type and H521A mutant MsHi-1. Catalytic constant (Kcat) refers to the number of substrate molecules converted to product per enzyme active site per unit time under saturating substrate conditions, while Michaelis constant (Km) represents the substrate concentration at which the reaction velocity reaches half of its maximum value. NS, not significant.
