Excavating the functionally crucial active-site residues of the DXS protein of Bacillus subtilis by exploring its closest homologues

Abstract

Abstract Background To achieve a high yield of terpenoid-based therapeutics, 1-deoxy-d-xylulose-5-phosphate (DXP) pathway has been significantly exploited for the production of downstream enzymes. The DXP synthase (DXS) enzyme, the initiator of this pathway, is pivotal for the convergence of carbon flux, and is computationally studied well for the industrially utilized generally regarded as safe (GRAS) bacterium Bacillus subtilis to decode its vital regions for aiding the construction of a functionally improved mutant library. Results For the 546 sequence dataset of DXS sequences, a representative set of 108 sequences is created, and it shows a significant evolutionary divergence across different species clubbed into 37 clades, whereas three clades are observed for the 76 sequence dataset of Bacillus subtilis. The DXS enzyme, sharing a statistically significant homology to transketolase, is shown to be evolutionarily too distant. By the mutual information-based co-evolutionary network and hotspot analysis, the most crucial loci within the active site are deciphered. The 650-residue representative structure displays a complete conservation of 114 loci, and only two co-evolving residues ASP154 and ILE371 are found to be the conserved ones. Lastly, P318D is predicted to be the top-ranked mutation causing the increase in the thermodynamic stability of 6OUW. Conclusion The study excavates the vital functional, phylogenetic, and conserved residues across the active site of the DXS protein, the key rate-limiting controller of the entire pathway. It would aid to computationally understand the evolutionary landscape of this industrially useful enzyme and would allow us to widen its substrate repertoire to increase the enzymatic yield of unnatural molecules for in vivo and in vitro applications.