Protein engineering enantioselectivity

The experimental evidences that medium engineering might represent an efficient method to modify or improve enzyme selectivity (alternative to protein engineering and to the time-consuming search for new catalysts) were immediately matched by the search for a sound rationale of this phenomenon. The different hypotheses formulated to try to rationalize the effects of the solvent on enzymatic enantioselectivity can be grouped into three different classes. The first hypothesis suggests that... 

A major advantage of enzymes as catalysts is that they are capable of inducing very high degrees of enantioselectivity and, consequently, they are particularly useful in the synthesis of enanhomerically pure compounds. In cases where the enanh-oselectivity is less than optimum it can generally be improved using protein engineering techniques such as in vitro evolution [10]. Hence, in this chapter we shall be mainly concerned with the application of enzymatic cascade processes to the... 

S-selectivity toward 1-phenylethanol [35]. Several other hydrolases have also been redesigned by protein engineering to invert or enhance their enantioselectivity [36-40]. 

Rotticci, D. Rotticci-Mulder, J.C. Denman, S. Norin, T. Hult, K. Improved enantioselectivity of a lipase by rational protein engineering. Chem-biochem. 2001, 2 (10), 766-770. 

I mproved Enantioselectivity of a Lipase by Rational Protein Engineering,... 

It is the most exciting and significant feature that the substrate specificity, enantioselectivity and regioselectivity can be profoundly affected by nature of solvents in which the enzyme molecule exists. This phenomenon has opened an alternative approach for changing specificity and selectivity of an enzyme other than both screening from nature and protein engineering in the field of synthetic organic chemistry. The ability of enzymes to discriminate... 

Since the reaction is taking place within the (chiral) active site of the enzyme, the reaction mechanism is stereospecific, the addition of H2 occurring with antistereochemistry. There is only one exception reported, the syn-hydrogenation of verbenone, carvone, and cyclohex-2-enone catalyzed by OYE of Nicotiana tabacum (Scheme 2.3) [12]. However, the aforementioned tremendous advances in molecular biology and biotechnology will facilitate recombinant expression and synthetic use of these novel biocatalysts by protein engineering. In this respect, recent examples already report structure-driven mutagenesis, which successfully improved reaction specificity or enantioselectivity [13,14]. 

Scheme 2.5c) [24,25], an activity that has potential in opiate biosynthesis. This strain has a morphine dehydrogenase that concomitantly reduces morphine into mor-phinone and has thus found applications in morphine detection. Finally, OYEs that reduce Baylis-Hillman adducts with complementary enantioselectivities have been either identified or generated by protein engineering (Scheme 2.5d) [26]. 

Rui, L., Cao, L., Chen, W., Reardon, K.F. and Wood, T.K. (2005) Protein engineering of epoxide hydrolase from Agrobacterium radiohacter ADI for enhanced activity and enantioselective production of (R)-l-phenylethane-l,2-diol. Appl. Environ. Microbiol., 71,3995-4003. 

Genetic engineering. The X-ray structures are known for many hydrolases, allowing for modeling of the substrate in the active site as well as structurally based, random or rational protein mutation to magnify or invert enantioselectivity. An example of the latter is provided by the rational design of a mutant of Candida antarctica lipase (CALB), which, instead of the wild-type R-selectivity, displayed... 

At the synthetic level we may expect increased emphasis on enantioselective catalysis usin metal complex catalysts as a key component of the manufacturing process (84). For biocatalysts there will unquestionably continue to be increasing interest in the "custom synthesis" of enzymes engineered for specific functions and conditions. The first example of the "ultimate" enzyme has been reported with the synthesis of the all-D form of HIV-1 protease (85-87). This enzyme exhibits a chiral specificity opposite to that of the naturally occurring L form and it may be generally predicted that enantiomeric proteins will exhibit reciprocal chiral specificity in all aspects of their interactions. These reciprocal chiral... 

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