Promiscuity substrate

Plant apocarotenoids have a wide variety of structures and functions. As expected, there is a small gene family of CCDs with different cleavage sites and somewhat promiscuous substrate selection. Some CCDs are stereo-specific, for example, 9-cis epoxycarotenoids are the substrates for NCEDs (9-cis expoxy dioxygenases) that produce the precursor of ABA biosynthesis, xanthoxin. Both linear carotenoids (lycopene) and cyclic carotenoids are substrates for cleavage at various double bonds including the central 15-15 and eccentric 5-6, 7-8, 9-10, 9 -10, and 11-12 bonds. Some CCDs cleave both linear and cyclic carotenoids and may cleave the same molecule twice, e.g., both 9-10 and 9 -10 positions. 

In comparison to the promiscuous substrate specificities of 6-OSTs, members of 3-OST family have distinctive substrate specificities. 3-OSTl synthesizes a characteristic disaccharide unit with the structure of GlcUA-GlcNS3S 6S (Figure 5), which is a critical modification step for synthesizing anticoagulant HS. 

The effects of neutral drift on promiscuous activities were explored in variants of bacteria P450 BM3 by Bloom etal Variants generated by error-prone PCR were screened to insure that they retained the ability to hydroxylate the substrate 12- -nitrophenoxydodecanoic acid at a level at least 75% that of the parental enzyme. Figure 16 shows the variability in the activity of 34 variants toward 12- -nitrophenoxydodecanoic acid and five alternative substrates. Even within a relatively short mutational distance of the parental enzyme, increases or decreases in activity toward various promiscuous substrates of up to fourfold were found. 

Furthermore, a useful way of assessing the magnitude of promiscuous activities is the rate acceleration iKsa/Kncm) or catalytic proficiency ( cat/- M/ uncat)- These parameters are indicative because they take into account the inherent reactivity of the substrate. In many cases, promiscuous activities occur, or are measured, with highly reactive substrates. Such activities are in a way expected. However, there are many cases in which promiscuous activities take place with substrates that are less activated than the native one. Examples include, the amidase activity of esterases such as lipases (Table 1, entry 7), the phosphodiesterase activities of P. diminuta PTE and alkaline phosphatase (Table 1, entries 10 and 8), and the PTE activities or various lactonases (Table 1, entries 11-13 and the notable fact that some of these lactonases do not hydrolyze the more activated aryl esters). In such cases, the chemical challenge posed by a less activated substrate is reflected in the more favorable comparisons of rate accelerations, or catalytic proficiencies, for the native versus the promiscuous substrates. 

It therefore seems that the very same active site can offer numerous modes of interactions, and some of these might be utilized by promiscuous substrates. It should be noted, however, that most of the above describes cases analyzed by kinetics and site-directed mutagenesis. Very few structures of the enzyme—substrate, or enzyme transition-state complexes, exist for both the native and promiscuous substrates. And thus, small, or even significant, changes in active-site configuration cannot be excluded in the described cases. 

As noted above, the magnitude of promiscuous activities varies over many orders-of-magnitude, both in absolute terms, and relative to the native activity (Section 8.03.4.3). The differences in reactivity between the native and promiscuous substrates can be manifested in differences in both and K. The schematic view is that the energetics of substrate binding are reflected in the Kyi, and catalysis by /feat- It is therefore expected that promiscuous substrates that bind poorly, due to steric hindrance, for example, will exhibit high Kyi values. However, many promiscuous substrates are characterized by low ifeat values. For example, a systematic analysis of >50 substrates for the enzyme PONl, the primary function of which is lipophilic lactonase, indicated that the promiscuous aryl ester, and phosphotriester, substrates all exhibit values in the millimolar range (0.8—5 mmol 1 ). This is despite the fact that their cat/ M values vary over three orders of magnitude. The differences in reactivity are therefore primarily due to eat values that vary by > 1000-fold. The probable... 

It is therefore anticipated, and often observed, that the mode of binding of the native substrate - that is typically mediated by several independent, enthalpy-driven interactions - is fundamentally different from that of the promiscuous substrates where hydrophobic and other entropy-driven interactions play a key role. 

Recent laboratory experiments followed the notion of a neutral drift by placing an enzyme under mutation and selection to maintain its native function. The data provide empirical evidence in support of the hypothesis that neutrality enables the formation of latent changes, or latent adaptation . It was found ° that latent evolutionary potentials are indeed very frequent within a neutral set of related enzyme mutants, and that these potentials are most often seen as changes in specificity for one or more promiscuous substrates. 

V. (2009) Promiscuous substrate binding explains the enzymatic stereo- and regiocontrolled synthesis of enantiopure hydroxy ketones and diols. Adv. Synth. Catal, 351, 1842-1848. 

The substrate selectivity of PsyA KSl has been previously examined using SNAC-thioesters, with acetyl-SNAC being shown as the preferred substrate, despite a rather promiscuous substrate profile (see Sect. 3.2.3). It was postulated that the use of the corresponding acyl-ACPs would represent a more realistic approximation of the native system. Moreover, de-acylation of the small ACP domain could be monitored by mass spectrometry. The assay consisted of incubation of PsyA... 

Kurina-Sanz, M., Bisogno, F.R., Lavandera, I., Orden, A.A., and Gotor, V. (2009) Promiscuous substrate binding explains the enzymatic stereo- and regiocontrolled synthesis of enantiopure hydroxy ketones and diols. Adv. Synth. Catal., 351 (11-12), 1842-1848. 

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