Mutagenesis application

M. Pohl, Protein design on pyruvate decarboxylase (PDC) by site-directed mutagenesis. Application to mechanistical investigations, and tailoring PDC for the use in organic synthesis. Adv. Biochem. Eng. BioUchnol. 1997, 58, 15-43. 

Enzyme promiscuity is clearly advantageous to chemists since it broadens the applicability of enzymes in chemical synthesis. New catalytic activities in existing enzymes can be enhanced by protein engineering - appropriate mutagenesis of the enzymes [106]. Some of the most illustrative examples of this unusual activity of common enzymes are presented below. 

During the last decade, significant advancements in biochemistry, molecular cloning, and random and site-directed mutagenesis, directed evolution of biocatalysts, metabolic engineering and fermentation technology have led us to devise methods to circumvent the disadvantages of whole-cell biotransformation discussed in Section 10.2. The applications of these methods are summarized in this section. 

Fig. 2.5. Possible applications of a coupling parameter, A, in free energy calculations, (a) and (b) correspond, respectively, to simple and coupled modifications of torsional degrees of freedom, involved in the study of conformational equilibria (c) represents an intramolecular, end-to-end reaction coordinate that may be used, for instance, to model the folding of a short peptide (d) symbolizes the alteration of selected nonbonded interactions to estimate relative free energies, in the spirit of site-directed mutagenesis experiments (e) is a simple distance separating chemical species that can be employed in potential of mean force (PMF) calculations and (f) corresponds to the annihilation of selected nonbonded interactions for the estimation of e.g., free energies of solvation. In the examples (a), (b), and (e), the coupling parameter, A, is not independent of the Cartesian coordinates, x. Appropriate metric tensor correction should be considered through a relevant transformation into generalized coordinates...

DsRed (Fig. 5.3E) is a bright RFP with excitation and emission maxima at 558 and 583 nm, respectively. Despite the bright red fluorescence, application of DsRed has been restricted, because of slow and inefficient maturation and its tetrameric structure [70, 71], The poor maturation efficiency has been overcome by random mutagenesis, which resulted in the fast maturing variant DsRedTl [72]. However, DsRedTl remains tetrameric. 

The introduction of techniques for mutagenesis by UV irradiation or by the use of chemicals considerably extended the applications of microbial studies to nutrition (Davis, 1954-1955). Auxotrophic mutants were produced with nutrient dependencies not shown in the untreated parental strains (Beadle and Tatum, 1940). The fortuitous discovery of penicillin by Fleming and its successful use in the treatment of infections (Florey) promoted exhaustive research into its mode of action. Eventually it was established that penicillin prevented the proliferation of gram-positive bacteria by blocking the synthesis of their cell walls... 

Objectives in the Application of Site-Directed Mutagenesis to the Study of Electron... 

Aside from these relatively direct applications of site-directed mutagenesis, combination of recombinant DNA techniques with other experimental strategies will no doubt prove to be of increasing importance. If the gene of interest can be expressed with sufficient efficiency in auxotrophs, then proteins in which selected amino acids are isotopically enriched [12] may be produced to increase the sensitivity and selectivity of magnetic resonance techniques. Alternatively, amino acid analogues that are recognized as substrates by aminoacyl tRNA synthetases may be incorporated randomly in place of the true substrate amino... 

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