Evolution of enzymes

Directed evolution of enzymes has been used to improve the reducing function of the enzymes. For example, this method was used to eliminate the cofactor requirement of B. stearothermophillus lactate dehydrogenase, which is activated in the presence of fructose 1,6-bisphosphate [12]. The activator is expensive and representative of the sort of cofactor complications that are undesirable in industrial processes. Three rounds of random mutagenesis and screening produced a mutant that is almost fully... 

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. 

Decarboxylases are one of the members of the enolase superfamily. The most important and interesting point of this class of enzymes is that they are mechanistically diverse and catalyze different overall reactions. However, each enzyme shares a partial reaction in which an active site base abstracts a proton to form a nucleophile. The intermediates are directed to different products in the different active sites of different members. However, some enzymes of this class exhibit catalytic promiscuity in their natural form. ° This fact is considered to be strongly related to the evolution of enzymes. Reflecting the similarity of the essential step of the total reaction, there are some successful examples of artificial-directed evolution of these enzymes to catalyze distinctly different chemical transformation. The changing of decarboxylase to racemase described in Section 2.5 is also one of these examples. 

The draggability of enzymes as targets reflects the evolution of enzyme structure to efficiently perform catalysis of chemical reactions, as discussed in the following section. 

Reetz, M.T. (2007) Directed evolution of enzymes for asymmetric syntheses, in Asymmetric Synthesis (eds Christmann M. and Braese S.), Wiley-VCH, pp. 207-211. 

Lutz, S. and Patrick, W.M. (2004) Novel methods for directed evolution of enzymes quality, not quantity. Current Opinion in Biotechnology, 15, 291-297. 

Zhao, H.M., Chockalingam, K. and Chen, Z.L. (2002) Directed evolution of enzymes and pathways for industrial biocatalysis. Current Opinion in Biotechnology, 13, 104—110. 

This chapter will consider some of the most interesting of current approaches to the evolution of enzyme mimics, in the context of continuing dramatic progress in protein and nucleotide engineering. There are excellent practical as well as intellectual reasons for the broad interest in this topic. Catalysis is a major preoccupation of the chemical industry if the application of the principles of biocatalysis can lead to robust and efficient catalysts tailor-made for reactions of economic importance the area will become even more a focus of intense activity and investment. 

Hartley, B. S. The Evolution of Enzymes. Plenary Lecture, Ninth International Congress of Biochemistry, Stockholm, Abstracts, 7 (1973). 

Turner, N., Directed evolution of enzymes for applied biocatalysis Trends BiotechnoL, 2003,21, 474-478 Johannes, T.W. and Zhao, H., Directed evolution of enzymes and biosynthetic pathways. Curr. Opin. Microbiol., 2006, 9, 261-267. 

Bershtein, S. and Tawfik, D.S., Advances in laboratory evolution of enzymes. Curr. Opin. Chem. Biol, 2008,12, 151-158. 

Alexeeva, M., Carr, R. and Turner, N.J., Directed evolution of enzymes new biocatalysts for asymmetric synthesis. Org. Biomol. Chem., 2003,1, 4133. 

Kuchner, O. and Arnold, F. H. (1997) Directed evolution of enzyme catalysts. Trends Biotechnol., 15, 523-30. 

In discussing enzyme action two different aspects should be considered i) catalysis, i.e., the ability to accelerate a given reaction and to govern its mechanism properly, and ii) specificity, i.e., the capacity to discriminate among several reagents. However, since the evolution of enzymes has been a process of selection there is a limit in such a specificity [12]. 

AMINOCYCLORRORANE-L-CARBOXYLATE OXIDASE ETIOCHOLANOLONE SULFATASE Evolution of enzyme catalysis,... 

However, this process in itself is not yet directed evolution, because only one cycle of mutagenesis/screening is involved. Isolated cases involving at least two such cycles began to appear, being the first cases of true directed evolution of enzymes (29c), but it was not until the period of 1993—1997 that the idea of directed... 

Albery, W.J. and Knowles, J.R. (1976) Evolution of enzyme function and the development of catalytic efficiency. Biochem., 15, 5631-5640. 

Babbitt, P.C. Gerlt, J.A. (1997) Understanding enzyme super-families chemistry as the fundamental determinant in the evolution of new catalytic activities. J. Biol. Chem. 27, 30,591-30,594. An interesting description of the evolution of enzymes with different catalytic specificities, and the use of a limited repertoire of protein structural motifs. 

A recent discovery in biochemistry is that RNA can act as an enzyme in chemical reactions, usually reactions involving RNA hydrolysis. Discuss the features of RNA structure that might favor evolution of enzymes composed entirely of a single polyribonucleotide chain, and describe a proposed mechanism for RNA-catalyzed hydrolysis of RNA molecules. 

Figure 11.3 Evolution of enzymes with random mutagenesis and DNA shuffling (adapted from Arnold, 1996).

U. T. Bornscheuer, Directed evolution of enzymes for biocatalytic applications, Biocatal. Biotransf. 2001, 19, 85-97. 

Retained substrate specificity (binding), changed chemistry A new enzyme evolves to supply substrate from an available precursor by evolution of enzyme using the substrate. The underlying hypothesis states that metabolic pathways evolve backwards A > 15 > C, 1 A >Pi is new, B >C is old enzyme ... 

Although the structure of haptens exerts an important influence on catalysis by antibodies, stabilization of the transition state by catalytic antibodies cannot always and cannot fully explain all observations. Sometimes, in analogy to the evolution of enzyme function, antibodies can take mechanistically unexpected detours. As an example, the corresponding antibody catalyzes the hydrolysis of arylamides according to Figure 18.3 by a factor of 2.5 x 105 over background. 

Verzhbinskaya, N.A. (1968). Biochemical evolution of enzyme systems as the base of functional evolution of vertebrate animals (In Russian). In Abiogenesis and Primary Stages of the Evolution of Life (A.I. Oparin ed.), pp.169-180, Nauka, Moscow. 

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