Structure-guided mutagenesis

In an another application, the enantioselectivity of carboxylesterase A (CesA) from Bacillus subtilis towards butyrate and caprilate esters of 1,2-O-isopropylideneglycerol was improved up to 13-fold by structure-guided mutagenesis. The mutagenesis of positions 166 and 182 in CesA yielded novel variants with enhanced enantioselectivity without significant loss of catalytic activity. ... 

Bhan, N, Cress, B.F., Linhardt, R.J., and Koffas, M, (2015) Expanding the chemical space of polyketides through structure-guided mutagenesis of Vitis vinifera stilbene synthase. Biochimie, 115, 136-143. 

Andreadeli A, Platis D, Tishkov V, Popov V, Labrou NE (2008) Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP. FEBS J 275 3859-3869... 

PDC enzymes usually prefer small aliphatic aldehydes or 2-ketoadd compounds as donors and aliphatic or aromatic aldehydes as acceptors, furnishing the corresponding acetoin and phenylacetyl carbinol derivatives [ 14). A number of PDC variants have been obtained by structure-guided directed mutagenesis with improved activity and stereoselectivity and the results have been summarized in previous reviews [6,14,48,49,56,61,62]. 

Structure-guided site-directed mutagenesis offers a great potential for the rapid preparation of mutants with new/improved activity when the structure of the enzyme and its catalytic mechanism are known [29,111-114]. 

Various isoforms of both HO and NOS can be expressed in recombinant systems. As a result, the immediate future will undoubtedly witness a wealth of mutagenesis experiments guided by the crystal structures. It also may be possible to trap in crystalline form the various intermediates of the HO reaction cycle, which will greatly facilitate a deeper understanding of the catalytic mechanism. Conformational dynamics appear to be quite important in HO, and hence, a variety of spectral probes such as NMR and fluorescence should prove especially useful in studying the role of protein dynamics in function. Overall there should be considerable optimism for understanding HO at the level of detail achieved for peroxidases and other well-studied enz5une systems. 

In cases in which a sharp question can be formulated and so suggest a test of a mechanism of solvent participation, site-directed mutagenesis provides an experimental tool, perhaps appropriately guided by computer simulation of the protein—solvent system. The participation of fixed or random (percolative) network structures in proton movement is... 

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