Enzyme catalytic promiscuity

Finally, it should be mentioned that recently a new type of enzyme catalytic promiscuity reaction has been reported, which does not involve any of the catalytic... 

Enzyme catalytic promiscuity, where enzymes catalyze not only reactions with their natural substrates but also with non-natural substrates, has begun to be recognized as a valuable research and synthesis tool [6-10, 12, 13]. In particular, the catalytic promiscuity displayed by sucrose-utilizing transglucosidases toward a wide range of acceptor molecules has started to be exploited to provide novel synthesis pathways that are currently not available. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

This model clearly shows that the catalytic machinery involves a dyad of histidine and aspartate together with the oxyanion hole. Hence, it does not involve serine, which is the key amino acid in the hydrolytic activity of lipases, and, together with aspartate and histidine, constitutes the active site catalytic triad. This has been confirmed by constructing a mutant in which serine was replaced with alanine (Serl05Ala), and finding that it catalyzes the Michael additions even more efficiently than the wild-type enzyme (an example of induced catalytic promiscuity ) [105]. 

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. 

Old and new enzymes developed by this mechanism are members of the same superfamily. The main champions of this mechanism are Gerlt and Babbitt (Gerlt, 1999 Babbitt, 2000). Readers are also encouraged to consult the reference on catalytic promiscuity by O Brien and Herschlag (O Brien, 1999). 

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). 

Recently, even examples of lipase-catalyzed Michael additions and aldol condensations have appeared [7]. These are dramatic examples of catalytic promiscuity, that is, the ability of an enzyme to catalyze more distinctly different chemical transformations [8], Often such activities are explained in terms of the active site offering a scaffold in which substrates adopt favorable conformations and/ or reactants are brought together in a desired geometry. Accordingly, after being observed in wild-type enzymes, these side activities can often be enhanced in site-directed variants, in which residues in or close to the active site are mutated. 

The potential of enzymes as practical catalysts is well described, and their activity and selectivity (stereo-, chemo-, and regioselectivity) for catalyzed reactions cover a broad range. Enzymes clearly constitute very powerful green tools for catalyzing synthetic chemical processes. In this context, the continuous increase of the market for enantiopure fine chemicals places enzymes as suitable catalysts for green synthetic processes. Catalytic promiscuity of enzymes in nonaqueous environments has been widely described and is related to the ability of a single active site to catalyze more than one chemical transformation for example, lipase B from Candida antarctica (CALB) is able to catalyze aldol additions, Michael-type additions, and so on [4]. 

This catalytic flexibility of enzymes is generally denoted as catalytic promiscuity [52-58], which is divided into substrate promiscuity (conversion of a nonnatural substrate), catalytic promiscuity (a nonnatural reaction is catalyzed), and cOTidition promiscuity (catalysis occurring in a nonnatural environment). Enzymes display three major types of selectivities ... 

Engineering Catalysis. The holy grail of enzyme redesign is the engineering of entirely new catalytic activities, a property which is often denoted as catalytic promiscuity [530,531]. The latter has been driven by the rapidly increasing number of crystal structures of proteins, which allow to understand the molecular details of their catalytic mechanism. In this context, it was possible to re-engineer the catalytic activities of well studied proteins to furnish switched activities or even completely novel functions, which are rarely found in Nature (Table 3.8). 

Table 3.8 Examples for the catalytic promiscuity of rationally designed enzymes...

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... 

Bomscheuer UT, Kazlauskas RJ. Catalytic promiscuity in biocatalysis using old enzymes to form new bonds and... 

Copley SD. Enzymes with extra talents moonlighting functions and catalytic promiscuity. Curr. Opin. Chem. Biol. 2003 7 265-272. 

Enzyme promiscuity is clearly advantageous to chemists since it broadens the applicability of enzymes in chemical synthesis. New catalytic activities in existing enzymes can be enhanced by protein engineering - appropriate mutagenesis of the enzymes [106]. Some of the most illustrative examples of this unusual activity of common enzymes are presented below. 

Even an entirely different enzyme can be changed to the one that has enolase activity. One representative example is the changing of a lipase to an aldolase utilizing the basicity of the catalytic triad via a simple mutation. The resulting promiscuous lipase has been demonstrated to catalyze the aldol reaction and Michael addition as shown in Fig. 23. 

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