Enzymatic promiscuity

The ability of enzymes to catalyze other reactions than those for which they have been evolved is called enzymatic promiscuity [99-102]. 

One of the striking features of enzymatic methylation is its exquisite regjoselec-tivity. For example, in the biosynthesis of novobiocin, a methyltransferase NovO catalyzes the methylation of only one of the three phenolic carbons and none of the three hydroxyl groups are methylated (Scheme 7.20). The exquisite regioselec-tivity is also compatible with substrate promiscuity. For example, the methyltransferase CouO from coumermycin A1 synthase catalyzes the methylation of both the mono- and bis-amides [75, 76]. 

Scheme 4.15 Examples of promiscuous enzymatic reactions conducted with the oxyanion hole of Candida antarctica lipase B (a) the aldol reaction [104] (b) the conjugate addition reaction (Michael addition) [105] (c) the epoxidation reaction [106],...

P. J. O Brien and D. Herschiag, Catalytic promiscuity and the evolution of new enzymatic activities, Chem. Bid. 1999, 6, R91-R105. 

Future BVMO redesign studies can exploit the localization of these hotspots by simultaneously mutating the respective residues that determine the plasticity of the substrate-binding pocket of BVMOs. In this context it is noteworthy that, for several mutations in CHMO, an extension of substrate acceptance was observed with concomitant high turnover to allow for preparative exploitation [56]. By this strategy, novel valuable BVMOs can be created that extend the catalytic potential of the presently available BVMOs and may ultimately combine substrate promiscuity with other enzymatic properties, for example thermal stability in case of PAMO. 

Drug distribution in such sites or compartments is a complex process that depends on the systemic circulation concentration and subsequent passage across single cell endothelial or epithelial membranes with specialized physical and molecular barrier functionality. For certain orally administered AIDS medications (e.g., zidovudine and didanosine), oral absorption is limited because of poor absorption from the G1 tract, enzymatic biotransformation in the intestinal epithelium, or first-pass effects (Sinko et al., 1995, 1997). For other AIDS drugs (e.g., protease inhibitors), oral absorption may be complete however, drug distribution into the brain is limited by drug efflux proteins, which promiscuously interact and translocate lipophilic substrates back into blood as they diffuse into the BBB endothelium (Edwards et al., 2005 Kim et al., 1998). 

The primary focus of this chapter is the role of promiscuity in the evolution of new enzyme functions. New enzymes have constantly emerged throughout the natural history of this planet. Over the past decades, enzymes that degrade synthetic chemicals were introduced to the biosystem, and enzymes associated with drug resistance, provide vivid examples of how rapid the evolution of new enzymatic functions can be. The first direct connection between protein evolution and promiscuity was made in 1976 by Jensen. In his landmark review, Jensen formalized the hypothesis that the starting points for evolution were provided by broad specificity, or promiscuity, of the ancestral enzymes. Jensen proposed that unlike modern enzymes that tend to specialize in one substrate and reaction, the primordial, ancient enzymes possessed very broad specificities, and thus few enzymes could perform many functions. Divergence of specialized enzymes, through duplication, mutation, and selection, led to the current diversity of enzymes, and to increased metabolic efficiency. 

This category refers to enzymes that catalyze different reactions (and not just different substrates) than the one they evolved for. As is the case with substrate and coenzyme ambiguity, the enzyme s activity with these alternative substrates is purely accidental, and is under no selection, and is therefore promiscuous by definition. As suggested, these cases include chemical transformations where the bonds that are broken, or formed, are different than those in the native substrate and reaction, and/or transformations that proceed through a different transition state. As discussed later, the promiscuous chemical transformations can be performed by the same catalytic side chains, and by essentially the same mechanism, as the native enzymatic function (Section 8.03.6). But there are also cases in which the enzyme utilizes different subsets of active-site residues, and somewhat different mechanisms, for the native and promiscuous functions (Section 8.03.6.1.4). 

In contrast to promiscuity that occurs within the same active site as the primary, native function, moonlighting relates to the utilization of protein parts outside the active site for other functions, mostly regulatory and structural,but sometimes enzymatic ones. Such activities can be recruited at later evolutionary stages, as indicated by the classical example of crystallins whereby metabolic enzymes were recruited later in evolution as structural components of eye lenses " (see Section 8.03.8.6.2 on gene sharing). 

We have initially described this trend in three different enzymes subjected to a selection for an increase in six different promiscuous activities. The same trend was identified in other laboratory experiments aimed at increasing promiscuous enzymatic and binding activities of various proteins (see Supplementary Table 8 in Aharoni Averaging eighteen cases in which data was provided for the effect of the selected mutations on... 

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