Below is more detailed description of a few gene/enzyme/reaction/pathway databases that are crucial to a metabolic reconstruction:
• Kyoto Encyclopedia of Genes and Genomes (KEGG): This is a
bioinformatics database containing
information on genes, proteins, reactions,
and pathways. The ‘KEGG Organisms’ section, which is divided into eukaryotesand prokaryotes, encompasses many organisms for which gene and DNAinformation can be searched by typing in the enzyme of choice. This resource can be extremely useful when building the association between metabolism enzymes, reactions and genes.
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• Gene DataBase (GeneDB): Similar to the KEGG resource, the Gene DataBase provides
access to genomes of various organisms. If a search for hexokinaseis carried out, genes for the organism of interest can be easily found. Moreover, the metabolic process
associated with the enzyme is also listed along with the information on the genes (in the case of hexokinase, the pathway is glycolysis). Therefore, with one click, it is very easy to access all the different genes that are associated with glycolysis. Furthermore, GeneDB has a hierarchical organizational structure for metabolism, and it is possible to see at what level of the chain one is currently working on. This helps broaden an understanding of the biological and chemical processes that are involved in the organism.
• BioCyc, EcoCyc and MetaCyc: BioCyc is a collection of over 200 pathway/genome
databases, containing whole databases dedicated to certain organisms. For example, EcoCyc which falls under the giant umbrella of BioCyc, is a highly detailed bioinformatics database on the genome and metabolic reconstruction of Escherichia Coli, including thorough descriptions of the various signaling pathways. The EcoCyc database can serve as a paradigm and model for any reconstruction. Additionally, MetaCyc, an encyclopedia of metabolic pathways, contains a wealth of information on metabolic reactions derived from over 600 different organisms.
• Pathway Tools [3]: This is a bioinformatics package that assists in the construction of pathway/genome databases such as EcoCyc (Francke et al.2005). Developed by Peter Karp and associates at the SRI International Bioinformatics Group, Pathway Tools comprises several separate units that work together to generate new pathway/genome databases. First, PathoLogic takes an annotated genome for an organism and infers probable metabolic pathways to produce a new pathway/genome database. This can be followed by application of the Pathway Hole Filler, which predicts likely genes to fill "holes" (missing steps) in predicted pathways. Afterward, the Pathway Tools Navigator and Editor functions let users visualize, analyze, access and update the database. Thus, using PathoLogic and encyclopedias like MetaCyc, an initial fast reconstruction can be developed automatically, and then using the other units of Pathway Tools, a very detailed manual update, curation and verification step can be carried out (SRI 2005).
• ENZYME: This is an enzyme nomenclaturedatabase (part of the ExPASY [4]
proteonomics server of the Swiss Institute of Bioinformatics). After searching for a particular enzyme on the database, this resource gives you the reaction that is catalyzed. Additionally, ENZYME has direct links to various other gene/enzyme/medical literature databases such as KEGG, BRENDA, PUBMED, and PUMA2 to name a few.
• BRENDA: A comprehensive enzyme database, BRENDA, allows you to search for an
enzyme by name or EC number. You can also search for an organism and find all the relevant enzyme information. Moreover, when an enzyme search is carried out, BRENDA provides a list of all organisms containing the particular enzyme of interest.
• PUBMED: This is an online library developed by the National Center for Biotechnology
Information, which contains a massive collection of medical journals. Using the link provided by ENZYME, the search can be directed towards the organism of interest, thus recovering literature on the enzyme and its use inside of the organism.
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Next steps of the reconstruction
After the initial stages of the reconstruction, a systematic verification is made in order to make sure no inconsistencies are present and that all the entries listed are correct and accurate (Francke et al.2005). Furthermore, previous literature can be researched in order to support any information obtained from one of the many metabolic reaction and genome databases. This provides an added level of assurance for the reconstruction that the enzyme and the reaction it catalyzes do actually occur in the organism.
Any new reactions not present in the databases need to be added to the reconstruction. The presence or absence of certain reactions of the metabolism will affect the amount of reactants/products that are present for other reactions within the particular pathway. This is because products in one reaction go on to become the reactants for another reaction, i.e. products of one reaction can combine with other proteins or compounds to form new proteins/compounds in the presence of different enzymes or catalysts(Francke et al.2005). Francke et al. (2005) provide an excellent example as to why the verification step of the project needs to be performed in significant detail. During a metabolic network reconstruction of Lactobacillus plantarum, the model showed that succinyl-CoA was one of the reactants for a reaction that was a part of the biosynthesis of methionine. However, an understanding of the physiology of the organism would have revealed that due to an incomplete tricarboxylic acid pathway, Lactobacillus plantarum does not actually produce succinyl-CoA, and the correct reactant for that part of the reaction was acetyl-CoA.
Therefore, systematic verification of the initial reconstruction will bring to light several inconsistencies that can adversely affect the final interpretation of the reconstruction, which is to accurately comprehend the molecular mechanisms of the organism. Furthermore, the simulation step also ensures that all the reactions present in the reconstruction are properly balanced. To sum up, a reconstruction that is fully accurate can lead to greater insight about understanding the functioning of the organism of interest (Francke et al.2005).