Promiscuous Enzymes

Further considerations include the physical and chemical properties of the fuel, its by-products, and any potential abihty to interfere with the cathodic chemistry. When thinking about the physical and chemical properties, researchers are usually interested in (1) whether the separator between the anode and the cathode is stable in the fuel and by-product solution, (2) the solubility of the fuel in water, (3) whether fuel evaporation will be an issue, and (4) the viscosity of the fuel. The first three points affect the actual amount of usable fuel, which further defines the energy density of the BFC, and the viscosity has an important effect on the mass transfer of the fuel to the electrode surface. Fuel tolerance is a special issue for BFC designs where fuel may enter the cathode half-cell. The cathodic enzyme, for example, could be denatured or inhibited by the presence of the fuel or its by-products. 

3 PROMISCUOUS ENZYMES VERSUS MULTIENZYME CASCADES VERSUS METABOLONS 

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In contrast to moonlighting proteins, which often are unknown to a researcher working in biocatalysis, proteins demonstrating catalytic promiscuity are fairly common and have also, in fact, been discussed in previous chapters of this book. Table 16.3 lists some examples of promiscuous enzymes, for a review, readers are referred to O Brien (1999). 

Hatano, 1995b) from Amycolaptosis sp. is known to catalyze more than one different chemical reaction using a substantially different substrate (Palmer et al., 1999). Investigation of this catalytic flexibility in the context of the enolase superfamily raises the question of whether this enzyme may represent an example of nature s present-day reengineering of the superfamily scaffold for an entirely new function. Other examples of catalytically promiscuous enzymes from other superfamilies have been observed, as reviewed by O Brien and Herschlag (O Brien and Her-schlag, 1999). 

Shelley Copley received her A.B. in Biochemistry and Ph.D. in Biophysics from Harvard University. After 3 years as a postdoc at MIT and the University of Colorado, she joined the Department of Chemistry and Biochemistry and the Cooperative Institute for Research in Environmental Sciences at the University of Colorado in 1990. She moved to the Department of Molecular, Cellular, and Developmental Biology in 2000. Her research focuses on the molecular evolution of catalysts and metabolic pathways, and particularly on the evolutionary potential of promiscuous enzyme activities. 

Table 3 Examples for directed evolution of promiscuous enzyme functions and their trade-offs with the native function ... 

Promiscuous enzyme, or promiscuous activity A promiscuous enzyme is one that has the ability to catalyze one or more reactions that are different from its primary physiological reaction. For example, a number of phosphatases also catalyze the hydrolysis of sulfate esters. 

S., and Las Heras-Vazquez, F.J. (2011) N-carbamoyl-P-alanine amidohydrolase from Agrobacterium tumefaciens C58 a promiscuous enzyme for the production of amino acids. J. Chromatogr. B Anal. Technd. Biomed. Life Sci., 879 (29), 3277-3282. 

The plant cell is a complex biochemical factory in which biosynthetic processes take place in different compartments. It is possible that the substrate for one enzyme will be produced in more than one cell compartment. In the case of promiscuous enzymes, two different potential substrates could be located in different cellular compartments. This can provide opportunities for bioengineering to form a new product or to increase the levels of current products. One can simply replace, add or remove an existing localization signal in order to modify the target compartment... 

Ding B, Schmeling S, Fuchs G (2008) Anaerobic metabolism of catechol by the denitrifying bacterium Thauera aromatica — a result of promiscuous enzymes and regulators. J Bacteriol 190 1620-1630... 

Brustad EM, Arnold FH (2011) Optimizing non-natural protein function with directed evolution. Curr Opin Chem Biol 15 201-210 Busto E, Gotor-Fernandez V, Gotor V (2010) Hydrolases catalytically promiscuous enzymes for non-conventional reactions in organic synthesis. Chem Soc Rev 39 4504-4523... 

Bridging the gap between organocatalysis and biocatalysis has been attained with the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) that has been shown to catalyse the Michael-type addition of acetaldehyde to nitrostyrene. The reaction proceeds in water, affords the (5)-configured product in 89% ee, and is believed to proceed via enamine formation that employs the amino-terminal proline of the enzyme. 2 0... 

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