Virtual Prediction of Zerumbone Compound in Lempuyang as Anti-Bacterial Agent Through In-Silico Approach

A native herbaceous plant of Southeast Asia, lempuyang is also widely distributed in Indonesia. This plant's primary bioactive component, Zerombone, has a variety of bioactivities, including an anti-bacterial effect. Typically, transglycosylase and alanine racemase were necessary proteins for forming peptidoglycan membranes and are now frequently used as anti-bacterial targets. Thus, using an in-silico approach, this work assessed the interactions between the zerumbone and the protein targets. The PubChem database was searched for the zerumbone compound (CID 5470187). Transglycosylase (PDB code: 1SLY), alanine racemase (PDB code: 4WR3), and the control protein target were also obtained from the PDB database . The Molegro Virtual Docker 5.0 version and the Discovery Studio application 21.1.1 versions were used to interface with and visualize the protein target and zerumbone compound. The result demonstrated that, like the control, the zerumbone compound in lempuyang blocks the active sites of the transglycosylase and the alanine racemase. The zerumbone-alanine racemase complexes additionally showed lower energy binding than other complexes. In conclusion, zerumbone in lempuyang can be a new candidate for anti-bacterial agents by inhibiting the synthesis of membrane peptidoglycan via suppressing transglycosylase and alanine racemase activities.


INTRODUCTION
As a tropical country, Indonesia is rich in mega-biodiversity, such as a medicinal plant.According to the Medicinal Herb Index, about 5,000 medicinal plants are found in Indonesia (Arozal et al., 2020).One of the medicinal plants found in Indonesia is lempuyang, scientifically known as Zingiber zerumbet, which is a native rhizomatous herbaceous from Southeast Asia and belongs to a member of the Zingiberaceae family (Yob et al., 2011).Based on the Statistics of Horticulture, the production of lempuyang in Indonesia reached 7219608 kg in 2022 (Irjayanti et al., 2022).Commonly in Indonesia, lempuyang and its extracts are utilized as a traditional herbal medicine (Padalia et al., 2018;Widyastiwi and Roseno, 2022), as a spice, as vegetables (Silalahi, 2018), or as ornamental plants.
According to Koga et al. (2016), terpenes and polyphenols are the primary chemical components found to dominate in lempuyang, with the main bioactive compound being zerumbone.Zerumbone is a valuable compound with various biological functions, including anti-bacterial capabilities (Veena et al., 2020;Ibáñez et al., 2023).Previous studies reported that the zerumbone compound has excellent anti-bacterial inhibitory properties in gramnegative and gram-positive microorganisms, such as Escherichia coli (Moreira Da Silva et al., 2018).The zerumbone compound in lempuyang exhibits anti-bacterial activity primarily through two main ways: imitation of anti-bacterial resistance mechanisms or chemical interference.Anti-bacterial agents frequently target the following processes in bacteria: (a) bacterial DNA replication and repair, (b) bacterial cell wall formation, (c) bacterial protein biosynthesis, (d) bacterial cell membrane destruction, or (e) metabolic pathway inhibition (Khameneh et al., 2019).Alanine racemase and transglycosylase are the two examples of proteins responsible for prokaryote cell wall biosynthesis (Sauvage et al., 2008;Wei et al., 2016;Martinez-Bond et al., 2022).
Alanine racemase is a PLP (pyridoxal-5′phosphate) enzyme that functions to catalyze the conversion of L-alanine to D-alanine, a crucial building block in the formation of the peptidoglycan layer in bacterial cell walls (Asojo et al., 2014;Wei et al., 2016).This enzyme is frequently used to develop new antibacterial candidates because it is abundant in all bacteria but absent in higher species (Azam and Jayaram, 2016).Moreover, transglycosylases, such as alanine racemase, are required for the polymerization of glycan strands and the formation of peptidoglycan, which is required for the structural integrity of the cell wall (Galley et al., 2014) and are now recognized as an intriguing target for anti-bacterial drugs and therapy (Chen et al., 2019;Martinez-Bond et al., 2022).As a result, as explained above, this work focused on the anti-bacterial activity of the zerumbone compound in lempuyang by triggering alanine racemase and transglycosylase utilizing in silico approach.

Ligand, Protein Structure Retrieval, and Binding Cavities Prediction
The 3-D structure of the zerumbone compound was downloaded from the PubChem National Center for Biotechnology Information (NCBI) database (https://pubchem.ncbi.nlm.nih.gov/) with CID 5470187.The target protein structures are transglycosylase (PDB code: 1SLY) (Chen et al., 2019) and alanine racemase (PDB code: 4WR3) (Soo et al., 2016) and downloaded from the PDB (Protein Data Bank) database (https://www.rcsb.org/).The protein target structures were predicted for their active sites using the Molegro Virtual Docker 5.0 version program with binding cavities parameters: van der Waals and maximum cavities of 5 (Bitencourt-Ferreira and de Azevedo, 2019; Bare et al., 2022;Sari et al., 2023).

Docking Simulation and Analysis
The active sites (binding cavities) of transglycosylase proteins were docked using the Molegro Virtual Docker 5.0 programs with specific protein grids, notably X = 12.77; Y = 49.69;Z = 49.06;Radius 13, and the protein grid docking of alanine racemase is X = 44.0;Y = 2.34; Z = 2.97; Radius 1.The docking parameters were Score Function Moldock Score [Grid], grid resolution of 0.30, algorithm MolDock SE, 10x number iterations, max iterations of 1500, max population size of 50, pose generation energy threshold of 100, tries of 10 -30, simplex evolution max steps of 300, neighbor distance factor of 1.00, multiple poses the number of poses of 5, energy threshold of 0.00, cluster similar poses RMSD threshold of 1.The protein target was docked with antibacterial control as a validation of the docking results of the zerombone compound, such as Bulgecin A (PDB code: 1SLY) for transglycosylase and Pyridoxal-5'-Phosphate (PDB code: 4WR3) for alanine racemase.Then, the docking results were analyzed by PyMol software version 2.2 and docking visualization to display 3D and 2D views and their interactions with the Discovery Studio program 21.1.1. version (de Azevedo Jr, 2019;Sari et al., 2021;Permatasari et al., 2022).

RESULTS AND DISCUSSION
Table 1 listed the interaction of transglycosylase as a receptor with the ligands, which are the zerumbone compound in lempuyang and bulgecin A as a control.The control interacted with the transglycosylase via SER487, MET498, THR501, TYR533, TYR552, GLU478, ASN553, GLY555, ALA554, and GLN496 amino acid residues through the hydrogen bonds, with low energy binding of -339.8 kJ/mol, as revealed in Table 1.Furthermore, the zerumbone compound in lempuyang also binds to the transglycosylase on the ILE497 amino acid residue by hydrogen bonds.Moreover, ALA491, MET498, ILE497, and TYR533 amino acid residues bind to zerumbone compounds via hydrophobic interactions.The binding energy of zerumbonetransglycosilase complexes is -207.6 kJ/mol (Table 1).Interestingly, the zerumbone compound in lempuyang and the control transglycosylase inhibitor (Bulgecin A) have the same active site interaction with the transglycosylase, as shown in Figure 1.Hydrogen Bonds

Carbon Hydrogen Bonds
The interaction between the zerumbone compound in lempuyang with the alanine racemase by the spending of energy binding of -243.4 kJ/mol (Table 2) amino acid residue via ALA163, ALA193, HIS159, TYR343, and TYR255 amino acid residues through seven hydrophobic interactions.Moreover, the alanine racemase control, the Pyridoxal-5'-Phosphate compound, interacted similarly with the zerumbone compound, as shown in Figure 2 and Table 2.The binding energy of Pyridoxal-5'-Phosphate-alanine racemase complexes was -288 kJ/mol, as listed in Table 2.The Pyridoxal-5'-Phosphate bound the alanine racemase via LYS34, ARG129, TYR38, TYR255, ARG280, TYR343, and PHE274 by hydrophobic interactions.Interestingly, the zerumbone compound and the control have the same amino acid residues in bounding of transglycosylase, which was TYR255 and TYR343, which indicated that the zerumbone compound in lempuyang had the same inhibition mechanism as the control, which is the Pyridoxal-5'-Phosphate.In earlier studies, Permatasari et al. (2022) also reported that the bioactive compounds of Syzygium myrtifolium interacted with alanine racemase via ALA193, TYR255, and TYR343.In addition, Soo et al. (2016) andThunnissen et al. (1995) identified TYR255 as the catalytic base.In this study, the zerombone compound also binds to TYR255, which means the zerumbone compound inhibits the alanine racemase in the catalytic base, indicating a high chance of interfering with bacterial cell wall biosynthesis.The binding energy of zerumbonealanine racemase complexes is lower than that of the zerumbone-transglycosylase complexes due to the bonds in zerumbone-alanine racemase dominated by hydrophobic interaction.Meanwhile, the zerumbonetransglycosylase complexes also had varied bonds, such as hydrogen bonds, hydrophobic interaction, and also unfavorable.Previous reports revealed that the number and interaction bonds affected the energy binding in proteinligand interaction (Bare et al., 2021;Sari et al., 2021).The hydrogen bonds, unfavorable bonds, and hydrophobic interactions contributed to the tightness and binding energy.The varied interaction of the ligands-proteins complex encouraged low binding energy and tighter interaction (Permatasari et al., 2022;Sari et al., 2023).

CONCLUSION
In summary, the alanine racemase and transglycosylase activities on the peptidoglycan cell wall were successfully inhibited by the zerumbone compound in lempuyang.In future research, molecular dynamics was enjoyable to perform.

Figure 1 .
Figure 1.Interaction between zerumbone compound and protein transglycosylase.(a) 3D view of zerumbone and Bulgecin A complexes with protein transglycosylase, (b-c).3D and 2D structures of the zerumbone-transglycosylase complex, and (d-e) 3D and 2D structures of the Bulgecin Atransglycosylase complex.The yellow ribbon structure represented transglycosylase, the pink stick structure represented Bulgecin A, and the blue stick structure represented the zerumbone compound.

Figure 2 .
Figure 2. The interaction between the zerumbone compound and the protein alanine racemase (a) 3D view of zerumbone and Pyridoxal-5'-Phosphate complexes with alanine racemase protein, (bc) 3D and 2D structures of the zerumbone-alanine racemase complex, and (d-e) 3D and 2D structures of the Pyridoxal-5'-Phosphatealanine racemase complex.The green ribbon structure represented the alanine racemase, the pink stick structure represented the Pyridoxal-5'-Phosphate, and the blue stick structure represented the zerumbone compound.

Table 1 .
Interaction between zerumbone compound and protein transglycosylase

Table 2 .
The interaction between the zerumbone compound and the alanine racemase protein