Domain replacement

Scheme 25 Example of thiolation (T) domains in type I NRPS. Nostocyclopeptide is shown as a representative nonribosomal peptide. Seven T domains are found in the seven modules, each depicted in a different color, responsible for the incorporation of the seven amino acid building block constituents of nostocyclopeptide. A reductase (R) domain replaces the more common TE domain for the formation of the unusual imine functionality.

Designer kinesins. Hybrids of kinesin have been prepared that consist of either (a) the sequence of conventional kinesin with the motor domain replaced by that of ncd or (b) the sequence of ncd with the motor domain replaced by that of conventional kinesin. In which direction along microtubules would you predict these hybrid kinesins to move ... 

The production of modified full-length derivatives of erythromycin has been achieved first by gene disruption experiments (see Section 12.3.1). Recent success in engineering new compounds via deletion of modules and domains, expression of single enzymes e.g., EryAI) and domain replacement have been described. 

Bedford, D., J. R. Jacobsen, G. Luo, D. E. Cane, and C. Khosla. (1996) A functional chimeric modular polyketide synthase generated via domain replacement. Chem. Biol. 3 827-831. 

Electron microscopic and chemical studies have led to the postulation of a low resolution model for the structure of the IgM pentamer [76,41]. From these studies and by comparison of IgM and IgG sequences a model can be proposed for the monomeric unit of IgM as shown in Fig. 11(a). The Fc portion of IgM (CM3 and C 4 domains) is very similar to that of IgG, with the non-paired C 3 domains resembling the CfL domains in having interposed N-linked, branched carbohydrate chains. The paired CM2 domains replace the hinge of IgG. There would, however, appear to be some potential for flexibility between the CM1 and CM2 domains and between C 2 and Cfi. Indeed, electron micrographs of pentameric IgM [4] indicate a monomer in which the F(ab )2 unit of Fig. 11(a) is rotated through 90° about its two-fold axis of symmetry (compare IgGl, Fig. 7). 

Fig. 11. The structure of IgM. (a) The monomeric unit. This schematic representation relies greatly on comparison of the aminoacid sequence of IgM (ju chain) and IgG (y chain) and extrapolation from known features of IgG structure. The Fab arms arc as for IgG, the paired CM2 domains replace the hinge, the CM3 domains are suggested to resemble the Cy2 domains in IgG, being unpaired with interposed carbohydrate, and the CM4 domains to resemble the paired C 3 domains of IgG. A disulphide bridge connects the heavy chains between the CM2 and CM3 domains. An additional feature is a tailpiece of 18 residues at the carboxy-termini of the heavy chains which may fold back across the CM4 domains. The molecular weight of the monomer is —19(11)0(1.

IX. ENGINEERED BIOSYNTHESIS OF NOVEL PEPTIDE ANTIBIOTICS BY TARGETED DOMAIN REPLACEMENTS... 

This targeted domain replacement of srf AC was successfully carried out for three bacterial domains of the grs operon activating Phe, Om. and Leu as well as for the Val-and Cys-activating domains of the fungal aevA gene. Surfactin derivatives produced by the chimeric synthetases were extracted from the cultured broth of different recombinant strains, examined for their hemolytic activity, and analyzed by infrared spectra and mass spectrometry. These studies clearly confirmed the identity of five novel Upopeptides that were produced by the selective domain exchange within the srf A operon. The surfactin isomers were found to carry the desired amino acid residue, as was expected from the corresponding domain replacement. 

Bedford D, Jacobsen JR, Luo G, Cane DE, Khosla C. A functional chimetk modular polydce-tide synthase generated via domain replacement. Chem Biol 1996 3 827—831. 

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