Structural Insights into the Enzymatic Activity of Cysteine Protease Bromelain of MD2 Pineapple

Razali, Rafida; Subbiah, Vijay Kumar; Budiman, Cahyo

doi:10.3923/pjbs.2020.829.838

Cited by 6 publications

(5 citation statements)

References 28 publications

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“…The competitive inhibition mechanism of hyptolide to Pk-ClpP is supported by the interaction pattern shown in Figure 9b,c, whereby most residues were involved in the interaction between Pk-ClpP and hyptolide or substrates are similar, with the uniqueness of more hydrophobic residues involved in a Pk-ClpP/hyptolide interaction. The involvement of more hydrophobic residues is often accompanied by more desolvation effects, which lead to stronger binding affinity to ligands [46,61,62]. Nevertheless, the ∆G values of Pk-ClpP with hyptolide or the substrate were found to be similar, which indicated that differences in the hydrophobic interaction minimally contributed to hyptolide binding stability.…”

Section: Discussionmentioning

confidence: 99%

Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide

et al. 2022

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The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue for antimalaria drugs discovery. The catalytic activity of the Clp system is modulated by its proteolytic subunit (ClpP), which belongs to the serine protease family member and is therefore extensively studied for further inhibitors development. Among many inhibitors, the group of β-lactone is known to be a specific inhibitor for ClpP. Nevertheless, other groups of lactones have never been studied. This study aims to characterize the catalytic properties of ClpP of Plasmodium knowlesi (Pk-ClpP) and the inhibition properties of a δ-lactone hyptolide against this protein. Accordingly, a codon-optimized synthetic gene encoding Pk-ClpP was expressed in Escherichia coli BL21(DE3) and purified under a single step of Ni2+-affinity chromatography, yielding a 2.20 mg from 1 L culture. Meanwhile, size-exclusion chromatography indicated that Pk-ClpP migrated primarily as homoheptameric with a size of 205 kDa. The specific activity of pure Pk-ClpP was 0.73 U µg−1, with a catalytic efficiency kcat/KM of 0.05 µM−1 s−1, with optimum temperature and pH of 50 °C and 7.0–7.5, respectively. Interestingly, hyptolide, a member of δ-lactone, was shown to inhibit Pk-ClpP with an IC50 value of 17.36 ± 1.44 nM. Structural homology modelling, secondary structure prediction, and far-UV CD spectra revealed that helical structures dominate this protein. In addition, the structural homology modeling showed that this protein forms a barrel-shaped homoheptamer. Docking simulation revealed that the inhibition was found to be a competitive inhibition in which hyptolide was able to dock into the catalytic site and block the substrate. The competitiveness of hyptolide is due to the higher binding affinity of this molecule than the substrate.

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Section: Discussionmentioning

confidence: 99%

Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide

et al. 2022

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“…All the data, including AI value and assay activities, were shown as a mean ± standard of deviation from three independent replications and descriptively analyzed (Razali et al 2020).…”

Section: Discussionmentioning

confidence: 99%

Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia

et al. 2022

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Abstract. Fazal BZ, Budiman C, Amin Z, Ling CMW. 2022. Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia. Biodiversitas 23: 2807-2815. Thermostable ?-amylases are being used in a wide range of industries, including food, textiles, detergents, pharmaceuticals, and fine chemicals. A good source of thermostable ?-amylases in thermophilic bacteria is found in high-temperature habitats like hot springs. Hence, this study aimed to screen, isolate, and characterize amylase-producing bacteria (APB) from the Poring hot spring in Sabah. Sediment and water samples were collected from the hot springs, serially diluted, plated onto the Luria Bertani agar medium containing starch, and incubated at 60°C for 48 hours. The amylase-producing bacterium was identified by the halo formation around the colony after the agar medium was stained with Lugol's solution. Nine colonies were found to be able to form halo zones, with a creamy colony (A7 strain) producing the highest amylolytic index (4.24). Further characterization of the A7 strain showed that the isolate was a Gram-positive, rod-shaped bacterium, with a positive reaction upon oxidase and catalase tests. The 16S rRNA sequence showed that the A7 strain had 99.81% similarity with the Anoxybacillus flavithermus, and therefore identified as A. flavithermus A7 strain. Further, the growth curve analysis indicated that the A7 strain grew well at 60°C. The 3, 5-dinitrosalicyclic acid (DNS) assay showed the crude enzyme secreted by the A7 strain exhibited optimum amylase activity at 60°C with 8.6 x 10-2 U/ml. This is the first APB obtained from hot springs in Sabah and promising for further studies and applications.

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“…A similar phenomenon was also reported for a small cysteine protease of pineapple MD2, harboring mobile catalytic site Cys at the loop region. Structural modeling of the Cys is quite distant from the ligand, yet it exhibits remarkable activity (Razali et al, 2020). Further, it was also known that the loop structure is highly flexible and mobile and the dynamic loop structure facilitates the catalytic activity of PAP (Hjörleifsson et al, 2021;Razali et al, 2020).…”

Section: Molecular Docking Of Papmentioning

confidence: 99%

“…Structural modeling of the Cys is quite distant from the ligand, yet it exhibits remarkable activity (Razali et al, 2020). Further, it was also known that the loop structure is highly flexible and mobile and the dynamic loop structure facilitates the catalytic activity of PAP (Hjörleifsson et al, 2021;Razali et al, 2020). Alternatively, a water molecule might mediate the interaction between the guanidinium group of Arg158 to PO4.…”

Section: Molecular Docking Of Papmentioning

confidence: 99%

Structural and Substrate Interaction Properties of Alkaline Phosphatase from Paenibacillus sp. PSB04: In-Silico Analysis

Lindang¹,

Budiman²

2022

OnLine Journal of Biological Sciences

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A Phosphate-Solubilising Bacterium (PSB) of Paenibacillus sp. PSB04 was previously isolated from the Sabah tropical rainforest in Malaysia. Interestingly, the genome sequence of the PSB04 strain harbored an Alkaline Phosphatase (AP) (EC 3.1.3.1) gene and was hypothesized to have unique structural characteristics. Therefore, this study aims to determine the AP three-Dimensional (3D) model and catalytic mechanism from Paenibacillus sp. PSB04 (PAP). To address this, the 3D model of this protein was built and docked into a model substrate of p-nitrophenyl phosphate. As a result, the best complex was shown to have the lowest binding energy of -5.9 kcal/moL. Furthermore, the complex showed the atomic coordination of catalytic residues of PAP and the substrate was similar to that of AP from Escherichia coli (ECAP), which implies that both APs shared a similar catalytic mechanism. In this mechanism, Ser 94 of PAP acted as nucleophilic residues, which were activated by the Zn ion. Arg 145 is predicted to be mobile due to its location in the loop region, which allows this residue to stabilize the substrate through direction or watermediated secondary interaction. Docking simulation of pNPP indicated that the putative residues involved in the catalysis mainly are Ser 94, Ser 141, Ala 146, Thr 147, Pro 148, Asp 293, and Glu 294. Glu 294 is considered a unique residue corresponding to Lys 328 ECAP, allowing the PAP to have a better affinity to stabilize the substrate in the binding cavity. Accordingly, a unique catalytic mechanism of PAP was proposed.

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Structural Insights into the Enzymatic Activity of Cysteine Protease Bromelain of MD2 Pineapple

Cited by 6 publications

References 28 publications

Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide

Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide

Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia

Structural and Substrate Interaction Properties of Alkaline Phosphatase from Paenibacillus sp. PSB04: In-Silico Analysis

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