Artificial enzymes evolution

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. 

This method of selecting catalytic sites significantly depends on spontaneous processes, in contrast to the development of artificial enzymes and catalytic antibodies. The selection process is based on self-assembly, selforganization and self-optimization. Therefore, this selection approach bears the characteristics of supramolecular chemistry. A similar concept is used in natural evolution processes, resulting in the complicated life forms we see around us today. Therefore, it is clear that we can design the self-organizational processes used in supramolecular chemistry to proceed according to the concepts followed by this natural evolutionary process. 

Klotz IM, Suh J. Evolution of synthetic polymers with enzyme-hke catalytic activities. In Artificial Enzymes. Breslow R, ed. 2005. Wiley-VCH. Weinheim, Germany, pp. 63-88. 

Nature provides us with the most efficient catalysts, enzymes that catalyze reactions under mild and green conditions (ie, atmospheric pressure, temperature, and aqueous solution). Efficiency and chano-, regio-, and stereoselectivity in enzyme-catalyzed reactions are so remarkable that they inspire scientists to design synthetic systems with comparable activity and selectivity [1,2], Enzymes made of proteins are more than just highly evolved catalysts. They recognize and respond to molecules other than their specific substrate and product [3]. The evolution of artificial enzymes is in its infancy and its main goal is efficient catalysis [1,3]. 

Further advancement of biocatalysis will require the use of directed evolution to bridge the functional gap between wild-type and desired biocatalyst properties. These studies underscore the power of directed evolution to create artificial enzymes derived from wild-type enzymes with the desired catalytic activity. Directed evolution techniques will continue to fulfill the promise of biocatalysis for industrial applications. 

A breakthrough in recombinant DNA technology and protein engineering was achieved by recognizing that the process of natural selection can be harnessed to evolve effective enzymes in artificial circumstances. In this framework of directed evolution , the processes of natural evolution for selecting proteins with the desired properties are accelerated in a test tube. The starting point is an enzyme with a measurable desired activity which still has to be improved. 

All these approaches have been used to alter protein function, to increase the activity or solubility of proteins, or to adapt enzymes for industrial applications. The goal of artificial man-made proteins with tailor-made activities is, however, still far away and none of the currently existing approaches provides the ultimate solution to the directed evolution of proteins. Nevertheless, numerous examples of successfully altered and improved proteins clearly show the power of directed evolution for protein design. 

Artificial chemical systems capable of Darwinian evolution have also been prepared from artificial laboratory genetic systems. Such systems were created in the laboratory by using an artificial DNA that contained six nucleotide letters rather than the four in standard terran DNA.6,7 These were chosen from the structures shown in Figure 4.1. The artificial systems can support the basic elements of Darwinian evolution (reproduction, mutation, and inheritance of mutated forms) even if the enzymes that support the evolution of artificial genetic systems are the natural terran enzymes that have evolved for billions of years to handle standard nucleobases. 

The counterpart of catalytic water oxidation to O, catalytic evolution via water reduction, is of equal importance in the context of water splitting (artificial photosynthesis) [15]. Innature, hydrogenase enzymes show excellent catalytic rates and efficiencies and can catalyze both proton reduction and oxidation. Consequently, several biomi-metic complexes have been developed which show excellent catalytic activity towards electrochemical production from water. 

Interestingly, the arginine switch mechanism was first recognized when it was artificially induced in AAT. When AAT was mutated in six distinct positions, a substantial increase of activity with aromatic substrates was observed. The crystal structure of the engineered enzyme showed that the aromatic side chains could be accommodated at the active site as a result of R292 movement. A similar observation was made on an AAT mutant form whose substrate specificity was broadened using direct evolution techniques, in order to include branched chain and aromatic amino acids. 

In all the examples studied to date, during half-transaminations, physiological or artificial , the medium derived proton has been shown experimentally to enter the Si face at C-4 of the coenzyme. This surprising stereochemical consistency in diverse types of pyridoxal-P-dependent enzymes has been interpreted by Dunathan and Voet [111] as evidence for the evolution of this family of enzymes from a common ancestor whose distribution of binding groups at the active site has remained essentially unaltered throughout the course of evolution. 

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