Yeast is a eukaryotic component that belongs to the fungi group. It is widely used as the expression system for the eukaryotic proteins. The report emphasizes on the acetyltransferase enzyme of the Baker's yeast that is widely used for the industrial processes. Alcohol O-acetyltransferase 1 is a catalytic enzyme used in the formation of esters. It is found in the cell membrane of the Saccharomyces cerevisiae commonly known as Baker's Yeast. It is a eukaryotic organism belong to the fungi group. As enzymes have optimum temperature and pH where they are most stable, the enzyme is most stable at 25⁰C at a pH range of 7-8 and its activity decreases if used at different pH and temperatures. It is found that the protein is active against the straight-chain carbon substrates as compared with the branched substrate with the same carbon number. The chemical reaction catalyzed by the enzyme is Acetyl Coenzyme A + Alcoholà Coenzyme A + Acetyl Ester. The enzyme belongs to the strain ATCC 204508 / S288c of Baker's Yeast. The report considers the various bioinformatic tools to identify its structure and functions. The bioinformatics tools are online software tools that have saved complete databases of the organisms and their proteins. The various tools used in this report in the context of alcohol O-acetyltransferase 1 are UniProt, NCBI, SWISS-PROT, and I-TASSER. The complete information of the enzyme is saved in NCBI and UniProt databases while the structural information of the enzyme can be elucidated from the PDB (protein database) source. The length of the acetyltransferase 1 enzyme is 525 amino acid long with a mass of 61,036 Dalton. The functional structure of the enzyme destabilizes as soon as it senses a different temperature and pH range because the amino acids lose structural stability. The structure of the protein is necessary to carry out the catalytic reactions. There are two domains of the enzyme one is from 24-41 and the other is 508-525 that are essential for the association of lipid droplets and endoplasmic reticulum. The protein has sequence similarity with one another protein named acetyltransferase 2. Moreover, the accession number of the enzyme to link it with other databases is P40353 and the databases are integrated to take the user to other databases for more information about the protein. The accession number is the unique number assigned to the proteins to ensure there is no redundancy in the accession numbers that can make the whole research process futile and waste. The activity of the enzyme is blocked by some potent inhibitors such as copper, zinc, cadmium, mercury cations, sulfhydryl agents, and unsaturated fatty acids. These metals and non-metals interfere with the functioning of the protein therefore, they are considered as an inhibitor. However, the amount required for interfering with the process is dependent on the sensitivity of the protein towards the inhibitor. The biotechnological use of it has shown in UniProt is that enzymes are commonly used in agriculture, brewing and winemaking, pest control, and renewable fuel. It is greatly used in the distillation industry due to its effect on the flavor of the wines. It maintains the fruitier characters of the drink for longer after bottling. Acetate esters formed from the action of acetyltransferase 1 are used in beverages and fermented foods because of their aroma. It is an anaerobic enzyme therefore, expressed in the absence of air and unsaturated fatty acids and aeration will repress its expression. The transcription of the enzyme is repressed by ROX1 and its binding site on the promoter of the enzyme is 5'-CCTATTGTTTT-3'. The results of this report are concluded using the bioinformatic databases. The structural information of the acetyltransferase 1 was obtained using the SwissProt and PDB. The secondary structures in the protein are available with their active sites. The report, therefore, provides a brief of how the information can be derived and used to check the existence of sequence similarity with other proteins of organisms of the same or different kingdom. The report also includes the literature of review that identifies the researches that incurred acetyltransferase. The extensive research of the protein is done to provide alternatives to the enzyme as it is not sustainable for the biofuel industry. Therefore, Eat1 protein is isolated from another yeast.
Alcohol o-acetyltransferase-1 is a catalytic enzyme of the Saccharomyces cerevisiae. It belongs to the fungi group and commonly known as Baker's yeast. The use of the enzyme is in the reaction where is transfers the acetyl group on the alcohol to form acetate ethers, fruity smelled substances. It is widely used in the agriculture industry, distillation industries, etc. The fruity smell of the wines and scents is due to the presence of these ethers formed by the acetyltransferase enzyme. The production of ethyl acetate using acetyltransferase enzyme was not sustainable therefore, a lot of work has been done to identify new proteins that resemble the function of acetyltransferase. Eat1 is one such novel enzyme whose action in catalyzing the transfer of the acetyl group on alcohol is similar to the action of acetyltransferase. It was more sustainable and isolated from the yeast named Wickerhamomyces anomalous. This used bioinformatics tools to identify the structure of the Eat1 enzyme by isolating it from the parent organism and culturing in vitro. Later, it was sequenced to identify the sequence similarity between the two enzymes (Kruis et al, 2017). The possibility of the Eat1 protein to be so effective in the catalysis reaction is due to its structural and sequence resemblance with the acetyltransferase enzyme. Furthermore, another difference between the two is the location. By performing the green fluorescent experiment where Eat1 was conjugated with green fluorescence chromophore to enable its tracking in the yeast. It was identified that Eat1 is mitochondrial proteins as it is located and functions in the mitochondria. However, acetyltransferase-1 is located in the membrane of the endoplasmic reticulum of the yeast cells. Therefore, the acetate ether by acetyltransferase occurs in the endoplasmic reticulum while the Eat1 associated catalysis of alcohol and acetyl coenzyme A occurs in the mitochondria of the yeast cells.
Scientists have researched extensively to sequence the model of Saccharomyces cerevisiae and the whole genome of the organism was sequenced using various databases and bioinformatic tools. It was, however, studied more deeply to update the annotation of the organism. The scientists updated the annotations by sequencing the single colony of the yeast to eradicate the errors of the result. Therefore, it acts as an anchor based on which further research is carried out (McIlwain et al, 2016).
The localization of the acetyltransferase enzyme was identified along with importance in the lipid droplet association. The N and C terminals of the two domains of the proteins, 24-41 and 508-525 are predicted as amphipathic helices. The scientists conducted the research and identified that the presence of these amphipathic helices is essential for the association between lipid droplets and endoplasmic reticulum. The experiment was performed by truncating these sequences of ATF-1 protein and observed that there occurred no association between lipid droplets and endoplasmic reticulum (Zhu et al, 2015).
Joaquin et al (2014) confirmed that the presence of the atf1 gene in the Saccharomyces Cerevisiae is necessary for the production of volatile acetate ether. The scientists made a mutant yeast that lacked the presence of the atf1 gene and named it atf-1. The production level of the ether in the mutant was significantly low and it was confirmed that the major function of the acetyltransferase was in the production of the ethyl acetate that has a fragrance and fruity smell, therefore, used in the production of the wines and bread (Christiaens et al, 2014).
The protein in the cell has active sites and they bind with effective promoters for increased expression in the cell. The promoter binding sites on the protein are present in the 5' end of the nucleotide sequence of the gene. It was identified that the protein has a binding site for promoter PKG1p at the 5' end. The binding of the promoter with the gene sequence of the acetyltransferase enzyme increased the production of the acetate ether in the culture from 25.04 mg/l to 78.76 mg/l and increased the aroma of the Chinese liquor. The entry of this research is in the UniProt database to provide the functional annotation of the gene and its interaction with the promoter sequence (Dong et al, 2014).
Many proteins interact with acetyltransferase-1 however, their action may vary. Certain proteins enhance the expression of the protein while others reduce the expression. For example, the PKG1p promoter binds with the acetyltransferase, and its expression increases. On the other hand, the genes BAT1 and IAH1, if deleted, increases the expression of the protein. They both inhibit the production of the acetyltransferase therefore, it was identified that the combined action of these three genes where PKG1p is inducing the expression and BAT1 and IAH1 when deleted, increases the expression of the atf1 gene (Li et al, 2017). The interacting genes are identified and they are removed so that they cannot inhibit the production of acetyltransferase to be used in the industries such as to scents, biofuels, agriculture, etc. In this researches, the major role was of the bioinformatic tools where the interacting partners of the protein are identified. The use of the acetyltransferase has interested bioengineering researches very much as it is considered as it can be used as an important biofuel. Acetyltransferases are potential enzymes in the formation of the fatty acid acyl esters by condensing the acyl or acetyl coenzymes with alcohol in the plants and yeast. Yeast is a suitable host for the production of the acetyltransferase compared with the E.coli. Therefore, the effective host is considered Yeast for the formation of the biofuel due to the binding of the PAGp1 promoter of acetyltransferase. These particular annotations can be retrieved from databases like UniProt where all the basic information of the protein is defined. These bioinformatics tools aim to provide a foundation before conducting specific research of the organism. The sequence annotation is the most important because the sequence of the protein is compared with other proteins. The common information can be applied which saves time and efforts of the researchers and the cost of the research.
The bioinformatics tools are online databases that collectively keep the complete information of the sequences of all the organisms that are sequenced to date. They are assigned a specific accession number so that identifying them in different databases would become easier. The UniProt database is online software that keeps the total information of the sequence, the amino acids that form the protein, various functions of the protein, and so on. Alcohol O-acetyltransferase 1 of the Saccharomyces cerevisiae was identified using these databases. For the first section, UniProt was used where the complete information of the sequence was available. The accession number of the protein in UniProt is P40353. This is specific for the enzyme acetyltransferase and the name of the gene for the enzyme is ATF-1. To use UniProt, the name of the gene or the accession number is entered in the search box and the whole information is on the page. The functions of the protein, its various domains, and the interactive sites of the protein. By using the UniProt tool, the location of the protein is also identified. Moreover, it provides an interconnection between the citations and other databases so that the user can procure other information also. The tool is easy to use and understand, therefore, widely used for the identification of the sequence information of the proteins of different organisms. The use of BLAST is followed by the UniProt and in BLAST, the sequences are compared to analyze the similarities among the sequences. In this report, after identifying the basic information from the UniProt, BLAST was run to identify the similar sequences that resemble acetyltransferase. BLAST stands for basic local alignment sequence tool and it calculates and measures the statistical significance of the similarity sequences. BLAST was carried for section two in which the acetyltransferase was run against two databases that are UniProtKB/SWISS-PROT and Protein Databank. BLAST shows the sequences homology of acetyltransferase with other proteins. However, the similarity percent of the sequence may vary. The BLAST tool also shows that the query ID of the enzyme is P40353.2 and this ID number is unique as the accession number of the enzyme. The method is not used solely for the identification of the protein but also for the determination of the nucleotides and amino acid sequences.
For completing session 4, the Swiss model was used where the homology modeling of the 3-dimensional structure of the protein was carried out. It is an effective method used to generate a three-dimensional structure for the given protein. Search using the Swiss-model tool involves four steps where the target sequence is first identified, aligned with the template, the structure is developed and model quality is assessed using the QMEAN. QMEAN is a statistical potential of the mean force. Swiss- model can be run using the database site expasy or using DeepView. After the Swiss- model, I-TASER was used to identify that if the sequence of the acetyltransferase has any conserved sequences in it. Modeling of the protein by I-TASSER is done by acquiring data from the protein database.
UniProt is the bioinformatic database that provides all the information on the protein. It involves the sequence information, FASTA sequence of the protein, functions, location, domain, etc. of the protein. Acetyltransferase is a catalytic protein and catalyzes the reaction by donating the acetyl group to the alcohol. The resulting acetate esters are fruity in smell, therefore, majorly used in the wine industries and agriculture. To use this tool, start with opening the UniProt webpage. The name of the protein is entered in the search box and all related names are suggested. The targeted protein of the Baker's yeast is selected and its curated information is viewed. The unique accession number of the protein makes the search process easier. The tool provides information on the structure of the protein and the domains. Furthermore, the amino acid sequence of the protein is also shown. The accession number of acetyltransferase-1 was P40353 and the protein has 525 amino acids. The protein was isolated from Saccharomyces cerevisiae commonly known as Baker's yeast (strain ATCC 204508 / S288c). The expression of the protein occurs in anaerobic conditions and aerobic environment blocks its expression. The results showed that the location of the enzyme is in the membrane of the endoplasmic reticulum. The results of the search are shown below.
After entering the name of the gene in the search box, this page appears where the similarly named proteins also appear. After choosing the desired protein, acetyltransferase in this case. After clicking on the desired sequence, it will direct the user to the main page of the protein where all the information will be annotated such as the functions of the proteins, sequences, etc.
This passage is taken from the UniProt database of the acetyltransferase and it is given that the major function of the acetyltransferase is synthesized esters of acetate from acetyl coenzyme A. The alcohols that accept the acetate group to form the esters are isoamyl acetate, ethyl acetate, butyl acetate, and so on. The enzyme cannot use long carbon chain alcohol. It can only use chins 10-12 carbon long and it is more effective for the unbranched chains of the alcohols.
Furthermore, the kinetics of the protein are also given which ensures the conditions where the protein will be most active and stable. Km is the substrate concentration required for the acetyl-coenzyme for the effective activity of the acetyltransferase. It can be seen in the diagram that the suitable temperature and pH for the effective functioning of the protein acetyltransferase are 25 degrees and 8 respectively. 8 pH is generally basic which shows that the protein functions best when the lower basic pH is provided in the culture. The importance of providing the pH and temperature in the specific range because the structure of the protein deforms when the suitable temperature and pH are not provided. The reason for providing the kinetic information of the protein is so that the user can list all the important conditions required for isolating acetyltransferase and using it in the industrial process. Moreover, if the required temperature and pH are not provided in the industrial process then the product may also become toxic. Therefore, it is of utmost importance that while experimenting, we provide the required conditions.
The taxonomy is the way to categorize the protein in specific categories according to the function of the protein whether it is an oxidase enzyme, transferase enzyme, and so on. It makes the searching and identification of the protein easy. Therefore, the categorization of the acetyltransferase is in the transferase category of the enzyme. This shows that the role of the protein or enzyme is to transfer the functional group from one substance to the other. The protein transfers the acetyl group on the alcohols to form the acetate esters.
It is an online tool or software that predicts the structure of the protein based on the algorithms of I-TASSER. It allows the prediction of the high-quality 3-D structure of the protein. The amino acid sequence of the acyltransferase is submitted in the I-TASSER and the template proteins that have similar folds are retrieved from the protein database (PDB) library by a meta-threading approach known as LOMETS. Later, the fragments from the protein database are assembled together to form a model of the protein. If the protein database fails to identify the templates by LOMET, then I-TASSER will predict a structure using the lower free-energy states. The predicted structures using I-TASSER are given below in the diagrams. All the predicted structures are based on the FASTA sequence of the acetyltransferase protein sequence. It is submitted to acquire the most similar structures. The top 10 threading templates used by I-TASSER are 3fotA, 2xhgA, etc. The templates of 3fotA, 2xhgA, and others are identified from the SWISS-PROT model. In the predicted sequences, the similarity of the sequences is highlighted with the colored sequences while others are in black. The software I-TASSER uses various programs to identify the top ten threading sequences. After identifying the top 10 similar sequences, the top five similar structures are predicted with the software and the C-score measures the confidence of the predicted structures. The structures in the databases are identified and predicted using the X-ray crystallography and the crystal lattice of the secondary structure of the proteins is submitted in these databases.
These are the top ten sequences that resemble the protein and are identified by the LOMETS library of the protein database (PDB). The library considers the templates and then predicts the structure of the protein. However, when the protein cannot predict the structure, I-TASSER does the function by predicting similar structures for the protein. In the aligned sequences, the colored sequences are those sequences that have similarity with the sequence while the remaining sequences in black are those that do not align with the given sequence of the protein.
It can be concluded from the above report that bioinformatics tools are the important software that is used for the identification of the protein sequence similarity and the structural similarity. The protein acetyltransferase is the catalytic enzyme used in the formation of the acetate esters. These esters are fruity smelled components and they can be used in the agriculture industry, brewing, winemaking, biofuel production, etc. The protein was run in different databases to get thorough information on the protein. The literature review of the protein shows that it has been widely used in the research process where the researchers use the interacting sites to increase the expression of the protein for industrial use. The protein expression is blocked by the presence of the air therefore, it is cultured in the anaerobic conditions in the culture to increase its efficiency. The UniProt databases gave all this information as it is updated whenever there is new information about the protein. After the UniProt, the BLAST was run for the protein using the UniProtKB/ SWISS-MODEL databases to identify the structural similarities of the protein and it was identified that the protein has some percent of t-similarity with acetyltransferase-2 of Saccharomyces cerevisiae and the chromosomal protein POC5 of the Xenopus laevis. Further, after running BLAST, the SWISS-MODEL was run and out of the top 10 most similar structures, three were shown in the report. The model is prepared by aligning the target protein with the template to identify the structural similarity. The similarity of all the templates was differentiated by coloring the nucleotides while the unmatched part of the proteins was left black for profound differentiation. The prediction of the similar structures was done using the I-TASSER tool of the bioinformatics. Therefore, it can be concluded that the protein shares its similarity with some proteins that can be visualized in the bioinformatic databases. This report also concludes that the importance of the tools is for the researchers as they do not have to invest their cost in the processes such as the sequencing of the protein therefore, saves time and money.
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