Protein engineering dehydrogenase

Bocanegra, J. H. A.,Scrutton,N. S. Perham, R. N. (1993). Creation of an NADP-dependent pyruvate dehydrogenase multienzyme complex by protein engineering. Biochemistry, 32, 2737-40. 

RD Chen, A Greer, AD Dean. Structural constraints in protein engineering the coenzyme specificity of Escherichia coli isocitrate dehydrogenase. Eur J Biochem 250 578-582, 1997. 

Protein engineering has been carried out to redesign substrate specificity of lactate dehydrogenase from Bacillus stearothermophilus (Wilks et al., 1988 Wilks et ah, 1990) ... 

Jongejan, A., Jongejan, J. A., and Duine, J. A., 1998, Homology model of quinohaemoprotein alcohol dehydrogenase from Comomonas testosteroni. Protein Engineering 11 1859198. 

Structurally, NADP differs from NAD only by a phosphate group esterified at the 2 C of the adenosine ribose, a difference which is reflected in the enzymatic roles NAD-dependent dehydrogenases are mostly involved in catabolic reactions, while NADP-specific enzymes are usually confined to biosynthetic pathways (1). The marked specificities displayed by dehydrogenases towards NAD and NADP have provided attractive model systems to understand the process of molecular recognition by protein engineering. 

The use of an LeuDH as an amino acid dehydrogenase showed a high L-enan-tiospecificity [24]. In this connection, an L-leucine dehydrogenase from Bacillus sphaericus has been applied very efficiently. The FDH from Candida boidinii is the preferred formate dehydrogenase for this process. The stability of this enzyme, which is available in technical quantities, has been remarkably improved by protein engineering and directed evolution [25], In particular the replacement of cys-... 

Figure 15-23. Recycling of NADPH with protein engineered formate dehydrogenase P2>.

Tishkov VI, Popov VO (2006) Protein engineering of formate dehydrogenase. Biomol Eng 23 89-110... 

Chemical modifications of mesophilic enzymes have met with more success than protein engineering. The strategies for stabilization have been reviewed [e.g. 39, 56] and include immobilization and hydrophilization. Chemical modification of trypsin and chymotrypsin gives artificial hydrophilization of non-polar areas of the protein surface resulting in marked thermostabilization and little loss of enzyme activity [57-59]. This phenomenon has also been demonstrated for Bacillus stearothermophilus lactate dehydrogenase by Wigley et al. [60] who used site-... 

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]. 

Yuhashi N, Tomiyama M, Okuda J, Igarashi S, Ikebukuro K, Sode K. Development of a novel glucose enzyme fuel cell system employing protein engineered PQQ glucose dehydrogenase. Biosens Bioelectron 2005 20 2145-2150. 

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