Identification of six important amino acid residues of MenA from Bacillus subtilis natto for enzyme activity and formation of menaquinone

Hu, Liu-xiu; Feng, Jingjing; Wu, Jiang; Li, Wei; Gningue, Sokhna mbacke; Yang, Ziming; Zhou, Wang; Liu, Yan; Xue, Zhenglian

doi:10.1016/j.enzmictec.2020.109583

Cited by 11 publications

(10 citation statements)

References 26 publications

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“…Menaquinone-specific isochorismate synthase (MenF), 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid synthase (MenD), 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (MenH), O-succinylbenzoic acid-CoA ligase (MenE), MenA, and MenG expression were increased 0.75-, 2.64-, 0.85-, 0.47-, 3.08-, and 4.53-fold, respectively. It has been reported that MenD is the only enzyme that adds the intermediate thiamine pyrophosphate to the secondary substrate β-C ( Dawson et al, 2008 ), the 1,4-dihydroxy-2-naphthoate octaprenyltransferase encoded by menA , polyisoprene pyrophosphate is used as the side chain to catalyze the formation of the water-soluble naphthalene compound 1,4-dihydroxy-2-naphthonic acid into membrane-bound 2-demethyl-menthoquinone, the precursor of MK ( Suvarna et al, 1998 ; Hu et al, 2020 ). The results showed that enhancing the precursor supply and eliminating the by-product synthesis pathway were common methods to improve strain performance.…”

Section: Resultsmentioning

confidence: 99%

Effects of Alkali Stress on the Growth and Menaquinone-7 Metabolism of Bacillus subtilis natto

et al. 2022

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Menaquinone-7 (MK-7) is an important vitamin K2, synthesized from the menaquinone parent ring and seven isoprene side chains. Presently, the synthesis of MK-7 stimulated by environmental stress primarily focuses on oxygen stress, while the effect of alkali stress is rarely studied. Therefore, this study researched the effects of alkali stress on the fermentation performance and gene expression of Bacillus subtilis natto. The organism’s growth characteristics, biomass, sporogenesis, MK-7 biosynthesis, and gene expression were analyzed. After a pH 8.5 stress adaptation treatment for 0.5 h and subsequent fermentation at pH 8.5, which promoted the growth of the strain and inhibited the spore formation rate. In addition, biomass was significantly increased (P < 0.05). The conversion rate of glycerol to MK-7 was 1.68 times higher than that of the control group, and the yield of MK-7 increased to 2.10 times. Transcriptomic analysis showed that the MK-7 high-yielding strain had enhanced carbon source utilization, increased glycerol and pyruvate metabolism, enhanced the Embden-Meyerhof pathway (EMP), tricarboxylic acid (TCA) circulation flux, and terpenoid biosynthesis pathway, and promoted the accumulation of acetyl-CoA, the side-chain precursor of isoprene. At the same time, the up-regulation of transketolase increased the metabolic flux of the pentose phosphate (HMP) pathway, which was conducive to the accumulation of D-erythrose 4-phosphate, the precursor of the menadione parent ring. This study’s results contribute to a better understanding of the effects of environmental stress on MK-7 fermentation by Bacillus subtilis natto and the molecular regulatory mechanism of MK-7 biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Effects of Alkali Stress on the Growth and Menaquinone-7 Metabolism of Bacillus subtilis natto

et al. 2022

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“…The computational methods aid a swift characterization and estimation of the functional properties of protein sequences. The physicochemical properties, viz., pI value, extinction coefficient, molecular weight, average hydrophobicity, and instability index are estimated through the ProtParam server for the representative sequences of set A and set B to determine their uniqueness (Table 1 ), as has been recently shown [ 48 ]. Isoelectric point or pI is the pH where the protein molecule has no net charge.…”

Section: Resultsmentioning

confidence: 99%

“…A most crucial step of semi-rational directed evolution strategy is the selection of hotspot loci whose mutations lead to a significant improvement of the catalytic/biological activity of the proteins [ 48 ]. Hotspots are the sites where alanine mutations lead to an increase of at least 2 kcal/mol in the binding free energy.…”

Section: Methodsmentioning

confidence: 99%

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

Runthala

Sai

Kamjula

et al. 2020

Journal of Genetic Engineering and Biotechnology

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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.

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“…The accuracy of a rationally evolved enzyme molecule is dependent on the identification of the hotspot residues, whose mutations could enhance its catalytic activity (Hu et al 2020). To analyze the hotspot regions, proximal to the active site and tunnel, and appropriately map the mutational landscape of the predicted protein molecule, the hotspot server 3.1 (Sumbalova et al 2018) is used.…”

Section: Theoretical Calculationsmentioning

confidence: 99%

NudF-boosted strategy to improve the yield of DXS pathway

Prasanna

Runthala

Shantier

2022

Preprint

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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.

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Identification of six important amino acid residues of MenA from Bacillus subtilis natto for enzyme activity and formation of menaquinone

Cited by 11 publications

References 26 publications

Effects of Alkali Stress on the Growth and Menaquinone-7 Metabolism of Bacillus subtilis natto

Effects of Alkali Stress on the Growth and Menaquinone-7 Metabolism of Bacillus subtilis natto

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

NudF-boosted strategy to improve the yield of DXS pathway

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