Probabilistic divergence of a template-based modelling methodology from the ideal protocol

Runthala, Ashish

doi:10.1007/s00894-020-04640-w

Cited by 3 publications

(2 citation statements)

References 94 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As the B . subtilis representative sequence CUB50584.1 is still not experimentally determined, its tertiary structure is modelled through template-based modelling methodology using MODELLER, as per the recently published strategy (Runthala 2021; Runthala and Chowdhury, 2019). For reliably predicting the model, the wild-type nucleoside diphosphate sugar hydrolase from Bdellovibrio bacteriovorus (PDB ID: 5C7Q) is used as the template, sharing a sequence identity of 36%.…”

Section: Theoretical Calculationsmentioning

confidence: 99%

NudF-boosted strategy to improve the yield of DXS pathway

Prasanna

Runthala

Shantier

2022

Preprint

Self Cite

View full text Add to dashboard Cite

Background: Terpenoids form a large pool of highly diverse organic compounds possessing several economically important properties, including nutritional, aromatic, and pharmacological properties. The 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 safe (GRAS) bacterium. The study aims to analyze the evolutionary conservation across the active site, and map the key residues for mutagenesis studies. The study would allow us customize the metabolic load towards the synthesis of prenol or isoprenol or any of the downstream molecules. Results: The 37-sequence dataset, extracted from 103 Bacillus subtilis entries, show a high phylogenetic divergence, and only six one-motif sequences ASB92783.1, ASB69297.1, ASB56714.1, AOR97677.1, AOL97023.1, and OAZ71765.1 show monophyly relationship, unlike a complete polyphyly relationship between the other 31 three-motif sequences. Further, only 47 of 179 residues of the representative sequence CUB50584.1 are observed to be significantly conserved. Docking analysis shows a preferential bias of ADP-ribose pyrophosphatase towards IPP, and a nearly 3-fold energetic difference is observed between IPP and DMAPP. Computational saturation mutagenesis of the seven hotspot residues identifies two key positions LYS78 and PHE116, encoded within loop1 and loop7, majorly interact with the ligands DMAPP and IPP, and their mutants K78I/K78L and PHE116D/PHE116E are found to stabilize the overall conformation. The loops are hereby shown to play a regulatory role in guiding the promiscuity of NudF towards a specific ligand. Conclusion: The study map the phylogenetic relationship between the 37 representative B.subtiis NudF sequences, and through sequence conservation, structural contact map, topological flexibility, and saturation mutagenesis of the active site residues, the essential residues regulating the interaction of NudF with IPP/DMAPP are deciphered. The study robustly screens its mutational landscape and localizes the two crucial residues LYS78 and PHE116 for directing the mutagenesis studies. The preliminary docking and simulation results also suggest a preferential bias of ADP-ribose pyrophosphatase towards IPP over DMAPP. The findings would pave the way for the development of novel enzyme variants with highly improved catalytic ability for the large-scale bioproduction of specific terpenoids with significant neutraceutical or commercial value.

show abstract

Section: Theoretical Calculationsmentioning

confidence: 99%

NudF-boosted strategy to improve the yield of DXS pathway

Prasanna

Runthala

Shantier

2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…As the B. subtilis representative sequence CUB50584.1 is still not experimentally determined, its tertiary structure is modeled through template-based modeling methodology using MODELER, as per the recently published strategy. , For reliably predicting the model, the wild-type nucleoside diphosphate sugar hydrolase from Bdellovibrio bacteriovorus (PDB ID: 5C7Q) is used as the template, sharing a sequence identity of 36%. As the first predicted protein model usually has several nonphysical local clashes, the constructed model is energetically relaxed through the conservative refinement strategy of Galaxyrefine2 to maximally retain the topology extracted from the template.…”

Section: Theoretical Calculationsmentioning

confidence: 99%

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

Prasanna

Runthala

2022

ACS Omega

Self Cite

View full text Add to dashboard Cite

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.

show abstract