Electronic Theses and Dissertation

Fusobacterium nucleatum (F. nucleatum) merupakan bakteri Gram negatif obligat anaerob dan ditemukan pada rongga mulut sehat ataupun dengan penyakit. Keberadaan bakteri ini dihubungkan dengan beberapa penyakit pada manusia, misalnya kanker usus, angina, paru-paru dan abses ginekologi. Pada rongga mulut, F. nucleatum merupakan salah satu bakteri penyebab penyakit periodontal. Adanya penyakit periodontal dapat menyebabkan bakteri masuk ke sirkulasi darah sehingga F. nucleatum dapat berpindah dari tempat asalnya di rongga mulut menuju bagian tubuh yang lain. Saat proses terjadinya penyakit periodontal, Fusobacterium nucleatum berperan sebagai “bridge bacterium”, yaitu menjadi penghubung bagi bakteri yang dijumpai pada fase awal kolonisasi plak dengan bakteri yang dijumpai pada kolonisasi akhir plak. Pada tahap lanjut dari penyakit periodontal dapat terjadi kehilangan perlekatan, kehilangan tulang alveolar, bertambahnya kedalaman poket, mobiliti gigi hingga kehilangan gigi. Kehilangan gigi dapat mempengaruhi fungsi pengunyahan, estetis, rasa percaya diri dan kualitas hidup dari seseorang.
Fusobacterium nucleatum memiliki membran luar yang merupakan ciri khas bakteri Gram negatif. Dinding sel terdiri dari membran luar dan dalam (sitoplasma) yang dipisahkan oleh ruang periplasmik yang berisi lapisan peptidoglikan. Membran luar berfungsi sebagai saringan molekular dan berisi phospholipids, lipopolisakarida (LPS), lipoprotein dan protein. bakteri Fusobacerium nucleatum subsp. nucleatum ATCC 25586 diidentifikasi memiliki tiga protein yang sangat diperlukan untuk keberlangsungan hidupnya dan non homolog dengan semua protein yang ada pada manusia. Ketiga protein target ini berlokasi di membran sitoplasmik, dua protein bersifat larut air yaitu phosphoenolpyruvate protein phosphotransferase dan phosphotransferase system fructose-spesific IIABC component sedangkan protein UDP-N-acetylglucosamine 1-carboxyvinyltransferase merupakan protein trans-membran.
Penelitian ini bertujuan untuk menentukan struktur 3D dari protein phosphoenolpyruvate protein phosphotransferase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase dan phosphotransferase system fructose-spesific IIABC component dari Fusobacterium nucleatum subsp. nucleatum ATCC 25586 menggunakan AlphaFold2. Tujuan penentuan struktur protein adalah untuk menentukan residu yang berperan sebagai tempat pengikatan ligan (binding residues). Hasil penentuan struktur protein dari phosphoenolpyruvate protein phosphotransferase memperlihatkan residu asam amino memiliki nilai confidence pada kategori very high, high, low dan very low. Tetapi residu yang berperan sebagai tempat pengikatan ligan memiliki nilai confidence very high dan high. Phosphoenolpyruvate-protein phosphotransferase memiliki residu pengikatan substrat pada asam amino Arg303, Arg339, Glu438, Asn461, Asp462, Arg472, dan Cys509. Hampir semua residu asam amino pada struktur protein UDP-N-acetylglucosamine 1-carboxyvinyltransferase memiliki nilai very high. Residu pengikatan atau katalitik dari UDP-N-acetylglucosamine 1-carboxyvinyltransferase adalah Asn24, Arg94, Leu98, Arg123, Pro124, Val125, Asp126, Leu127, His128, Phe163, Gly167, dan Asp308. Struktur
phosphotransferase system fructose-specific IIABC component berdasarkan hasil pemodelan Alphafold 2 memperlihatkan bahwa tidak semua residu asam amino memiliki tingkat confidence yang tinggi, ada yang berada pada kategori rendah berdasarkan nilai predicted local distance difference test.
Tahapan selanjutnya dilakukan molecular docking dari 8364 senyawa yang terdapat pada database drugbank terhadap protein phosphoenolpyruvate protein phosphotransferase dan UDP-N-acetylglucosamine-1-carboxyvinyltransferase. Hasil penelitian didapat bahwa asam pteroat merupakan senyawa yang dapat menghambat protein phosphoenolpyruvate protein phosphotransferase dengan nilai ikatan afinitas sebesar -6,8. Sedangkan protein UDP-N-acetylglucosamine-1-carboxyvinyltransferase dapat dihambat oleh senyawa conivaptan dengan nilai ikatan afinitas sebesar -12,6.
Hasil penelitian secara in vitro memperlihatkan bahwa konsentrasi 12.5% merupakan konsentrasi hambat minimum dan konsentrasi 25% merupakan konsentrasi bunuh minimum dari senyawa asam pteroat terhadap bakteri F. nucleatum subsp. nucleatum ATCC 25586. Sedangkan konsentrasi 25% merupakan konsentrasi hambat minimum dan konsentrasi 50% merupakan konsentrasi bunuh minimum dari senyawa conivaptan terhadap bakteri F. nucleatum subsp. nucleatum ATCC 25586. Tidak terdapat perbedaan yang signifikan secara statistik antara kelompok asam pteroat dan kelompok conivaptan (p>0.05)
Kata kunci: Fusobacterium nucleatum, struktur protein, molecular docking

Fusobacterium nucleatum (F. nucleatum) is an obligate anaerobic Gram-negative bacterium and is found in healthy oral cavity or that with disease. The presence of these bacteria is associated with several diseases in humans, such as colon cancer, angina, lung, and gynecological abscesses. In the oral cavity, F. nucleatum is one of the bacteria that causes periodontal disease. The presence of periodontal disease can cause the bacteria to circulate through bloodstream so that F. nucleatum can move from its place of origin in the oral cavity to other parts of the body. During the development of periodontal disease, Fusobacterium nucleatum acts as a "bridge bacterium", which is a link for bacteria found in the early phase of plaque colonization with those in the final phase. Moreover, advanced stages of periodontal disease include the loss of attachment, loss of alveolar bone, increased pocket depth, and tooth mobility to tooth loss. Further, tooth loss can affect the masticatory function, aesthetics, self-confidence, and quality of life of a person. Fusobacterium nucleatum has an outer membrane that is characteristic of Gram-negative bacteria. The cell wall consists of an outer and inner membrane (cytoplasm) separated by a periplasmic space that contains a layer of peptidoglycan. The outer membrane serves as a molecular sieve and contains phospholipids, lipopolysaccharides (LPS), lipoproteins, and proteins. Fusobacterium nucleatum subsp. nucleatum ATCC 25586 was identified as having three proteins that are indispensable for its survival and are non-homologous to all proteins present in humans. These three proteins are located in the cytoplasmic membrane, where two of which are water-soluble, namely phosphoenolpyruvate protein phosphotransferase and phosphotransferase system fructose-specific IIABC component. One of the proteins is a trans-membrane protein, namely UDP-N-acetylglucosamine 1-carboxyvinyltransferase. This study aimed to determine the 3D structure of phosphoenolpyruvate protein phosphotransferase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase and phosphotransferase system fructose-specific IIABC component from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 using AlphaFold2. The purpose of determining protein structure is to determine the residues that act as binding residues. The results of determining the protein structure of phosphoenolpyruvate protein phosphotransferase showed that all amino acid residues have confident category in the very high, high, low and very low. Residues that act as ligand binding sites have confident category in the very high and high. Phosphoenolpyruvate-protein phosphotransferase has substrate binding residues on the amino acids Arg303, Arg339, Glu438, Asn461, Asp462, Arg472, and Cys509. Almost all amino acid residues in the protein structure of UDP-N-acetylglucosamine 1-carboxyvinyltransferase have very high values. The binding or catalytic residues of UDP-N-acetylglucosamine 1-carboxyvinyltransferase are Asn24, Arg94, Leu98, Arg123, Pro124, Val125, Asp126, Leu127, His128, Phe163, Gly167, and Asp308. The structure of phosphotransferase system fructose-specific IIABC component system based on the results of Alphafold2 modeling reveals that not all amino acid residues have a high level of confidence, some are in the low categories based on the predicted local distance difference test value. The next step was molecular docking of 8364 compounds contained in the drugbank database of phosphoenolpyruvate protein phosphotransferase and UDP-N-acetylglucosamine-1-carboxyvinyltransferase. The results of the study suggested the potential inhibitory activity of pteroic acid against phosphoenolpyruvate protein phosphotransferase with a binding affinity of -6.8 kcal/mol. The protein UDP-N-acetylglucosamine-1-carboxyvinyltransferase was shown to be potentially inhibited by conivaptan with a binding affinity of -12.6 kcal/mol. The results of in vitro studies show that a concentration of 12.5% is the minimum inhibitory concentration and a concentration of 25% is the minimum bactericidal concentration of pteroic acid compounds against F. nucleatum subsp. nucleatum ATCC 25586. While the concentration of 25% is the value of minimum inhibitory concentration and the concentration of 50% is the minimum bactericidal concentration value of the conivaptan compound against the bacterium F. nucleatum subsp. nucleatum ATCC 25586. There was no statistically significant difference between the pteroic acid group and the conivaptan group (p>0.05). Keywords: Fusobacterium nucleatum, protein structure, molecular docking