Engineering Enzyme Specificity

Carter, P., Wells, J.A. Engineering enzyme specificity by "substrate-assisted catalysis." Science 237 394-399, 1987. 

Lippow SM, Moon TS, Basu S, Yoon SH, Li X, Chapman BA, Robison K, Lipovsek D, Prather KL. (2010). Engineering enzyme specificity using computational design of a defined-sequence library. Chem Biol, 17, 1306-1315. 

Engineering Substrate Specificity. Although the serine proteases use a common catalytic mechanism, the enzymes have a wide variety of substrate specificities. For example, the natural variant subtiHsins of B. amyloliquefaciens (subtiHsin BPN J and B. licheniformis (subtiHsin Carlsberg) possess very similar stmctures and sequences where 86 of 275 amino acids are identical, but have different catalytic efficiencies, toward tetraamino acid -nitroanilide substrates (67). 

Lastly, with the goal of further testing enhancement of substrate specificity and reaction rate on a larger scale with respect to the number of samples, a novel engineered E. coli strain has been developed, termed SELECT (Aace, adhC, DE3), which requires acetaldehyde as C-source for growth and maintenance. The scheme constitutes one of the first cases where in-vivo selection has identified unnatural enzyme specificity. 

IDH displays a 7000-fold preference for NADP whereas IMDH displays a 100-fold preference for NAD (5-7). High resolution X-ray crystal structures have been obtained for both enzymes with and without coenzymes bound (3, 4, 8, 9). The kinetic and catalytic mechanisms have also been determined (5, 7). In addition, a large quantities of recombinant proteins are readily purified, facilitating biochemical and structural analyses. The strict specificity together with an extensive knowledge of their structure and biochemistry makes IDH and IMDH ideal targets for rationally engineering coenzyme specificity. 

Biological enzymes are well known to carry out epoxidation. For example, MMO is an efficient and selective catalyst for epoxidation of small terminal olefins such as ethylene, propylene, and 1-butene [246,247]. Lipases have been used to generate peroxoacids which in turn are used for epoxidation reactions [248,249]. The subject has been reviewed [250]. Biocatalytic systems are of interest not only because they can carry out enantioselective epoxidation of substrates, but also because they offer the exciting possibility of being engineered for specific transformations of nonnatural reagents. 

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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