In silico structural analysis of NDH-2 isoforms in Pseudolycoriella hygida (Diptera:Sciaridae)

Pseudolycoriella hygida is a sciarid species first identified in the Ribeirão Preto campus of the University of São Paulo and has been maintained under laboratory conditions since the 1960s. Psl. hygida, along with a few other sciarid species, has served as a non-conventional model organism in cytogenetics, biochemistry and genomics. Recently, our group characterized the mitochondrial alternative oxidase (AOX), a terminal oxidase that bypasses the O₂ reduction by complex IV, and we are currently investigating an alternative NADH dehydrogenase (NDH-2), which oxidizes NADH/FADH₂ and transfers electrons to the electron transport chain. From genomic data analysis, we identified a single ndh-2 gene in Psl. hygida that encodes at least four transcriptional isoforms. Two transcripts differ by 18 nucleotides due to an alternative splicing event, resulting in a six amino-acid difference near the N-terminus, adjacent to the conserved GXGXXG catalytic motif. In Rossmann-fold proteins, this motif is involved in binding NADH or FADH₂. Our in silico analyses indicate that both Psl. hygida NDH-2 isoforms present two catalytic domains but may differ in substrate affinity. To investigate whether both protein isoforms potentially maintain biochemical function, we predicted their structures using AlphaFold3 and compared them with the crystallographic structure of the alternative NADH dehydrogenase from Saccharomyces cerevisae (PDB: 4G73, 33% sequence identity) using ChimeraX. Structural superposition of the longer isoform (508 amino acids; RMSD=0.783 Å) showed close alignment of the catalytic sites with minor differences in the GXGXXG motif. In contrast, the shorter isoform (502 amino acids; RMSD 0.797 Å) displayed distortion of the first catalytic motif caused by the six-amino-acid deletion, displacing a tryptophan residue away from the FAD-binding region. Nevertheless, the second catalytic site is fully conserved in both isoforms and is likely capable of binding NADH efficiently. These results provide structural insights on Psl. hygida NDH-2 and indicate that the isoforms may differ in substrate affinity. Experimental characterization will be needed to validate the functional relevance of these structural differences.
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2024/23632-7) and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).