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Enzyme Coding Regions - Background Information

Chain number

All of the enzyme coding regions in the Registry consist of single polypeptide chains that can fold into catalytically active molecules. Many enzymes in living organisms are more complicated than this, consisting of two or more subunits that associate with one another to form the active protein. Engineering such enzymes into a chassis will require careful design since the subunits will be required in some fixed "stoichiometric" ratio -- such as 1:1, 2:1, 3:2, etc. That means that to avoid wasteful (and perhaps harmful) accumulation of an excess of one of the subunits, the expression of the separate coding sequences will need to be coordinated.

Chemical composition

Most enzymes are proteins -- polymers of amino acid residues -- but a very important small set of enzymes actually consists of RNA molecules. These RNA enzymes are called ribozymes. Although the Registry at present contains no ribozymes, that's likely to change since ribozymes have the important feature that they can be formed directly from DNA merely by transcription, and do not require translation into protein before they can exert their catalytic effects. That feature will surely soon be exploited by synthetic biologists. (Note that when ribozymes are included in the Registry, they will be classified under "RNA" parts.]

Classification by reaction type

The major classification scheme used for enzymes comes from the International Union of Biochemistry and Molecular Biology (http://www.chem.qmul.ac.uk/iubmb/enzyme/). This "Enzyme Commission" (or "EC")system is based upon the precise chemical reaction(s) catalyzed by the enzyme. These fall into six main classes: (1) oxidoreductases, (2) transferases, (3) hydrolases, (4) lyases, (5) isomerases, and (6) ligases. Each class is further subdivided into subclasses which in turn are broken down into sub-subclasses! Finally, each specific enzyme has an indexing number within its sub-subclass. As an example of how this system works, consider a popular synthetic-biology enzyme, luxI (acyl-homoserine-lactone synthase). It is a transferase in the third subclass acyltransferases and falls into the first sub-subclass "transferring groups other than amino-acyl groups." Within this sub-subclass it was the 184th enzyme to be categorized. Thus it gets an indexing ("EC") number 2.3.1.184. Note that since EC numbers refer to a large set of enzymes that catalyze the same reaction, when you describe an enzyme by its EC number, you also need to give its source if you expect others to repeat your work. Thus "luxI (EC 2.3.1.184)" is not an adequate description. The correct term in this case might be "luxI (EC 2.3.1.184) from Vibrio fischeri."

Use of EC numbers

Biochemists and molecular biologists seldom refer to enzymes with the full EC names and numbers except when publishing definitive papers where it's important that there be no confusion about exactly which enzyme they are talking about. Nevertheless, the EC numbers have grown in importance as the number of known enzymes has increased. Since the names of enzymes can often be confusing to non-specialists, the EC numbers play an important role in minimizing confusion. They also are convenient tags to use in database management. The EC system does have a drawback, however. Many enzymes have not yet been classified. Since the effort to do so is ongoing, with time this problem will almost certainly be overcome.

BRENDA

Work with enzymes involves many practical considerations such as the optimum pH, standard methods for activity assay, kinetic parameters etc. The database called BRENDA (subtitled "The Comprehensive Enzyme Information System") contains a wealth of such information easily located, particularly with the aid of EC numbers. BRENDA also has links to other protein, enzyme and metabolic pathway databases. Check it out at http://www.brenda-enzymes.info/index.php4.

Biochemical Pathways

Enzymes almost never act alone. They are linked into complex networks called "metabolic pathways." Such networks track the transformations of all the biomolecules in living cells. Classical biochemistry, starting in about 1900 focused attention upon small-molecule metabolism - for example, how glucose gets converted into ethanol in anaerobic yeast cultures or into lactate in muscle tissue during strenuous exercise. Half a century later, molecular biology began to decipher macromolecular metabolism - synthesis (and degradation) of polymers like DNA and RNA that make up the information storage and transmission systems of cells, and, most importantly from the point of view of synthetic biology, the synthesis and degradation of proteins. In projects involving metabolic enzymes synthetic biologists should understand how the enzymes they use may interact with chassis metabolism. A good way to approach that question is to learn how the enzymes fit into metabolic paths in unmodified cells. For this task, the premier database is KEGG (Kyoto Encyclopedia of Genes and Genomes). Other databases like SwissProt/UniProt and BRENDA may allow you to follow a link directly to the relevant part of KEGG. In the absence of that shortcut, you can go directly to KEGG at http://www.genome.jp/kegg/.