Computationally Decoding NudF Residues To Enhance the Yield of the DXP Pathway

[Image: see text] Terpenoids form a large pool of highly diverse organic compounds possessing several economically important properties, including nutritional, aromatic, and pharmacological properties. The 1-deoxy-d-xylulose 5-phosphate (DXP) pathway’s end enzyme, nuclear distribution protein (NudF), interacting with isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), is critical for the synthesis of isoprenol/prenol/downstream compounds. The enzyme is yet to be thoroughly investigated to increase the overall yield of terpenoids in the Bacillus subtilis, which is widely used in industry and is generally regarded as a safe (GRAS) bacterium. The study aims to analyze the evolutionary conservation across the active site for mapping the key residues for mutagenesis studies. The 37-sequence data set, extracted from 103 Bacillus subtilis entries, shows a high phylogenetic divergence, and only six one-motif sequences ASB92783.1, ASB69297.1, ASB56714.1, AOR97677.1, AOL97023.1, and OAZ71765.1 show a monophyly relationship, unlike a complete polyphyly relationship between the other 31 three-motif sequences. Furthermore, only 47 of 179 residues of the representative sequence CUB50584.1 are observed to be significantly conserved. Docking analysis suggests a preferential bias of adenosine diphosphate (ADP)-ribose pyrophosphatase toward IPP, and a nearly threefold energetic difference is observed between IPP and DMAPP. The loops are hereby shown to play a regulatory role in guiding the promiscuity of NudF toward a specific ligand. Computational saturation mutagenesis of the seven hotspot residues identifies two key positions LYS78 and PHE116, orderly encoded within loop1 and loop7, majorly interacting with the ligands DMAPP and IPP, and their mutants K78I/K78L and PHE116D/PHE116E are found to stabilize the overall conformation. Molecular dynamics analysis shows that the IPP complex is significantly more stable than the DMAPP complex, and the NudF structure is very unstable. Besides showing a promiscuous binding of NudF with ligands, the analysis suggests its rate-limiting nature. The study would allow us to customize the metabolic load toward the synthesis of any of the downstream molecules. The findings would pave the way for the development of catalytically improved NudF mutants for the large-scale production of specific terpenoids with significant nutraceutical or commercial value.