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

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.

Keywords: Coevolution; Consurf; DXS; Directed evolution; Motif; Phylogeny.

Fig. 1

The MEP pathway. The pathway produces IPP and DMAPP as the 5-carbon building blocks for the biosynthesis of isoprenoids. It condenses glyceraldehyde 3-phopshate and pyruvate through DXS and forms IPP and DMAPP through six added steps. DMAPP linkage with one or two molecules of IPP forms monoterpene or sesquiterpene, respectively

Fig. 2

Structural overlap of DXS structures of Escherichia coli and Deinococcus radiodurans, showing a structural conservation of 468 residues across the secondary structure elements. The structures are superimposed and represented through Chimera 1.14 [21]

Fig. 3

Algorithmic flowchart for the analysis from sequence dataset to functional annotation and coevolving residues across the active site

Fig. 4

Variation of residue frequencies between the representative sequences Q7VRH9.1 and Q8D357.1 and AJW87412.1 and WP_007410329.1 of the two datasets, belonging to Enterobacteriaceae and Bacillus species respectively. A substantial variation in the residue percentages is observed for a few residues within and between the two datasets

Fig. 5

PSIPRED based estimation of the secondary structure for the representative sequences (a) Q7VRH9.1, (b) Q8D357.1, (c) AJW87412.1, and (d) WP_007410329.1 of the two sets A and B

Fig. 6

Prediction of protein transmembrane structure for representative sequences Q7VRH9.1, and Q8D357.1, and AJW87412.1 and WP_007410329.1

Fig. 7

Phylogenetic tree of the 546 sequence dataset of DXP synthase. An average pairwise score of 1.012 within the range of 1.0175 to 2.876 is found

Fig. 8

Phylogenetic tree of 76 sequences of DXP synthase proteins in Bacillus subtilis. The 13 transketolase sequences are marked with blue squared boxes, and it shows a clear segregation of the two enzymes

Fig. 9

a Statistically significant occurrence of tandem motifs within the 63 DXS sequences of Bacillus subtilis, in correlation with the reference structure 6OUW. Motifs 1 and 3 are found statistically conserved across all homologs. b Conservation logo and statistical E value scores of localized motifs

Fig. 10

Schematic representation of the active site residues of the representative structure 6OUW. a Active site protein pocket yielded by CastP [45]. b Conservation profile of Consurf [46], maroon being the most conserved residue. c MI network [47] of the conserved coevolved residues encoded in 63 DXS sequences. a Labels outward of the second circle represent the residue loci, and the colored square boxes indicate the level of sequence conservation within the intensity range of low (blue) to high (red). The two internal circles show proximity mutual information and cumulative mutual information scores respectively. As per MISTIC protocol, the curved central linkers connect residues with statistically significant MI scores (> 6.5), with red, black, and gray orderly indicating the residue pairs with the top 5% scores, average scores between 70 and 95% and lowest scores. d Structural localization of the 10 conserved coevolving residues, indicating the most preserved for directed evolution methodologies