In vitro reconstitution

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

Pereira MM, Carita JN, Teixeira M. 1999. Membrane-bound electron transfer chain of the ther-mohalophilic bacterium Rhodothermus marinus Characterization of the iron- sulfur centers from the dehydrogenases and investigation of the high-potential iron- sulfur protein function by in vitro reconstitution of the respiratory chain. Biochemistry 38 1276. 

Aspinall-O Dea, M., M. Wentworth, A. Pascal, B. Robert, A. Ruban, and P. Horton. 2002. In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants. Proc. Natl. Acad. Sci. USA 99 16331-16335. 

KAHN, R.A., BAK, S., SVENDSEN, I., HALKIER, B.A., M0LLER, B.L., Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum, Plant Physiol., 1997,115, 1661-1670. 

Figure 4. In vitro reconstituted 30 nm chromatin fiber. Dynamic structural changes in the chromatin fiber in the absence (top) or presence (bottom) of linker histone HI with different NaCl concentration were observed by AFM. Nucleosomes were reconstituted on the 106 kb plasmid and then fixed in the buffer containing 50 mM (top left) or 100 mM NaCl (top right). Nucleosomes were well-spread in 50 mM NaCl but attached each other and partially aggregated in 100 mM NaCl. After the addition of histone HI, the thicker fibers were formed. The width of the fibers is 20nm in 50mM NaCl (bottom left) or 30 nm in lOOmM NaCl (bottom right)...

Hayes JJ, Lee KM (1997) In vitro reconstitution and analysis of mononucleosomes containing defined DNAs and proteins. Methods 12 2-9... 

Massanz, C., Schmidt, S. and Friedrich, B. (1998) Subforms and in vitro reconstitution of the NAD-reducing hydrogenase of Alcaligenes eutrophus. J. Bacterial., 180, 1023-9. 

Quadri LEN, Keating TA, Patel HM, Walsh CT (1999) Assembly of Pseudomonas aeruginosa Nonribosomal Peptide Siderophore Pyochelin in vitro Reconstitution of Aryl-4, 2-bisthiazoline Synthetase Activity from PchD, PchE, and PChF. Biochemistry 38 14941... 

Foisner, R., Leichtfried, F. E., Herrmann, H., Small, J. V., Lawson, D., and Wiche, G. (1988). Cytoskeleton-associated plectin in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins. /. Biol. Chem. 106, 723-733. 

Keywords Biocatalysis Biosynthesis In vitro reconstitution Natural products ... 

Fig. 13 (a) Three enzymes (EntE, EntB and EntF) are required for the in vitro reconstitution of 64. (b) Biosynthesis of 64 by the stepwise condensation of three ArCP-bound DHB groups (79) and three PCP-bound L-serines (80a). Enterobactin (64) chelates ferric iron with high affinity via the six phenolate hydroxyl groups... 

The oxidative mechanism of RNR R2 differs from that of MMOH in requiring an additional electron, since Tyrl22 provides only a single electron. This electron is needed to convert P to X. It has been shown that external reductants such as excess Fe(II) or ascorbate can provide this electron in in vitro reconstitution reactions [87,97], Since the diiron site is buried 10 A below the protein surface, a long-range electron transfer pathway is required to deliver this extra electron to the diiron center. Such a pathway involving a number of amino acid residues has been proposed from examining the crystal structure of R2 [98],... 

CW Carreras, C Khosla. Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase. Biochemistry 37 2084-2088, 1998. 

The next major step was the development of an assay to determine the functional identity of the deduced PDesat-TnAllZ desaturase. Initial efforts to develop an in vitro reconstitutive biochemical assay combined the recombinant PDesat-TnAllZ protein purified from E. coli with phospholipids and a biochemical fraction from beef liver containing cytochrome b5 reductase and cytochrome b5. These efforts were unsuccessful (Knipple and Roelofs, unpublished). 

Kuempel, D., Swartzendruber, D.C., Squier, C.A., and Wertz, P.W, In vitro reconstitution of stratum corneum lipid lamellae, Biochim. Biophys. Acta, 1372, 135, 1998. 

Chow, D., Ho, J., Nguyen Pham, T. L., Rosejohn, S., and Garcia, K. C. (2001b). In vitro reconstitution of recognition and activation complexes between interleukin-6 and gpl30. Biochemistry 40, 7593-7603. 

Another possibihty to gain the product encoded by a silent gene cluster can be achieved by a method called the in vitro reconstitution approach. A necessity is that the substrates of the pathway are predictable. The in vitro reconstitution of an entire biosynthetic pathway usually involves the separate overexpression of each gene, the purification of the resulting protein and the performance of enzymatic studies. Thus, the discovery of a fully elaborated metabolic product by this approach is likely to be very laborious. For example, after discovering a cryptic sesquiterpene synthase in the genome of S. coelicolor (64), the new epi-isozaene (8) could be produced successfully. This metabolite was shown to be an intermediate in the assembly line of the known Streptomyces sesquiterpene albaflavenone (9) (135). 

Gamier, M., Dimchev, A., Boujrad, N., Price, J., Must, N. and Papadopoulos, V. (1994) In vitro reconstitution of a functional peripheral-type benzodiazepine receptor from mouse Leydig tumor cells. Mol. Pharmacol, 45,201-11. 

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