Background (continued)


Figure 5 Tryptophan
Tryptophan Synthesis Pathway
Tryptophan (Trp), an aromatic amino acid (Figure 5), is needed to synthesize proteins and as a precursor to niacin, serotonin (a neurotransmitter), and melatonin. There are six steps involved in tryptophan synthesis; the enzymes manipulated in the synthesis of ivePRAI, PRAI and IGPS, catalyze steps three and four respectively (Garrett, 1999). Two examples of maps for this pathway can be found at Metabolic Pathways of Biochemistry and Genome Net.
PRAI (phosphoribosyl anthranilate isomerase) catalyzes the isomerization of N-(5'-Phosphoribosyl)-anthranilate to Enol-1-o-carboxyphenylamino-1-deoxyribulose phosphate (CdRP) through a "rearrangement of the ribulose moiety," as shown in Figure 6. (Garrett 887).
Figure 6 PRAI catalyzes the isomerization of PRA to CdRP.

IGPS (indole-3-phosphate synthase) catalyzes the synthesis of indole-3-phosphate by inducing ring closure and decarboxylation of CdRP, as shown in Figure 7.

More about these enzymes can be found here.
Figure 7 IGPS catalyzes the synthesis of the indole ring
DNA Recombination Methods

DNA shuffling is a recombination technique that does exactly what its name implies. Accurate prediction of tertiary structure and function of an enzyme from its amino acid sequence is uncertain, and therefore the creation of a new protein through synthesis and expression of a randomly sequenced gene is often futile. If segments of the original DNA are conserved and merely recombined in a different order (shuffled), then the preserved sequences should still encode for segments of the original structure. The resultant gene has a higher chance of encoding a functional enzyme (Harayama 1998, Crameri 1998).

Figure 8 General steps in DNA shuffling:
1. One gene or several homologous genes are amplified through PCR.
2. The DNA is then fragmented using DNAse or a restriction enzyme in order to create many short, random sequences.
3. These sequences are allowed to recombine at random, creating lengths of DNA that preserve large "chunks" of original sequencing. When these contain sequences from different homologous genes, they are known as chimeras.
also see: Directed Enzyme Evolution (HF Arnold)

PCR, or polymerase chain reaction, is used to amplify (make multiple copies of) a segment of DNA. First, doublestranded DNA is denatured -- the helix is unwound the strands are separated -- allowing an oligonucleotide primer to bind to each separate strand. The primers are extended, complementary to the target DNA, using Taq polymerase. Completion of replication yields two copiesof the original double-stranded DNA segment. This process is then as many as 45 times. That the number of copies of DNA doubles with each cycle of repetition (Klug p515). Random-primer PCR, a technique employed by Altamirano et al., enables the random replication of a target sequence. Other forms of PCR include error-prone PCR, which results in random mutagenesis, and "sexual" PCR (Harayama,1998). Both of the latter techniques may be used in DNA shuffling.

A helpful animation of the PCR process may be found here; the Weizmann Institute also has an excellent PCR website.

Gene Libraries Once mutated sequences of DNA have been created, gene libraries are used to "store" the sequences or to express or produce large amounts of the enzyme in question. This is accomplished by ligating the mutant DNA into bacterial or viral DNA -- the organism (vector) thus becomes a living "storehouse" for the cloned sequence. Later, the cloned sequence can be identified using a DNA probe and recovered if the vector is bacterial, or via plaque hybridization if the vector is viral (Klug p510-512).
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Structural Modification

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Nikki Jarrett, 11/20/00
Biochemistry 462bH, Dr. D. Bourque