Bacteria, protein structure-function studies

Recently, active recombinant a-LTX has been generated using bacteria in which both thioredoxin reductase and glutathione reductase are inactivated to improve the formation of disulphide bonds in expressed proteins (Li et al. 2005). The toxin is expressed as a fusion with glutathione-S-transferase (GST), which is used for affinity purification of the recombinant toxin and can be subsequently removed by selective proteolysis. Considering the relative ease of generating recombinant proteins in bacteria, this approach will facilitate structure-function studies of a-LTX. 

The tryptophan synthase bienzyme complex from enteric bacteria provides an important example wherein RSSF has been used to good advantage for the study of both enzyme mechanism and protein structure-function relationships. This enzyme complex is composed of heterologous a- and P2-subunits arranged in a nearly linear a-(3-(l-a array (81). The a-subunit catalyzes the aldolytic cleavage of IGP to indole and G3P, while the P-subunit catalyzes the PLP-dependent condensation of i-Ser and indole to yield i-Trp. The aP-reaction is essentially the sum of the individual a- and P-reactions (scheme I). Indole, the common intermediate produced at the a-site, is direcdy channeled to the P-active site via a tunnel located in the interior of the protein complex which directly interconnects the a- and P-catalytic centers (81-84). Although the individual subunits may be isolated and are functional, formation of the bienzyme complex not only increases the catalytic activities of the separate subunits by nearly 100-fold, but also alters the thermodynamic stability of P-site reaction intermediates and introduces heterotropic allosteric interactions between sites. 

---