Unnatural substrates

It is apparent that the use of enzymatic catalysis continues to grow Greater availabiUty of enzymes, development of new methodologies for thek utilization, investigation of enzymatic behavior in nonconventional environments, and the design and synthesis of new biocatalysts with altered selectivity and increased stabiUty are essential for the successhil development of this field. As more is learned about selectivity of enzymes toward unnatural substrates, the choice of an enzyme for a particular transformation will become easier to predict. It should simplify a search for an appropriate catalyst and help to estabhsh biocatalytic procedures as a usehil supplement to classical organic synthesis. 

Uroporphyrinogen I (16c), a constitutional isomer of uroporphyrinogen III, also plays no direct role in porphyrin and corrin biosynthesis, but this unnatural substrate is methylated to give 17c10c f in the presence of SAM by the methyl transferase of some bacteria. A constitutional type I dihydroisobacteriochlorin can be obtained by methylation of uroporphyrinogen I with methylase Ml. Methyltransferase M1 is able to methylate the unnatural precorrin once more to give the trimethylpyrrocorphin type I.IOc 1... 

Other interesting examples of proteases that exhibit promiscuous behavior are proline dipeptidase from Alteromonas sp. JD6.5, whose original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus [121, 122] and aminopeptidase P (AMPP) from E. coli, which is a prohne-specific peptidase that catalyzes the hydrolysis of N-terminal peptide bonds containing a proline residue [123, 124]. Both enzymes exhibit phosphotriesterase activity. This means that they are capable of catalyzing the reaction that does not exist in nature. It is of particular importance, since they can hydrolyze unnatural substrates - triesters of phosphoric acid and diesters of phosphonic acids - such as organophosphorus pesticides or organophosphoms warfare agents (Scheme 5.25) [125]. 

One drawback of biocatalysis is that enzymes are not available in both enantiomeric forms. Particularly where a class of enzymes whose natural substrates are optically active, such as nucleosides, it can be difficult if not impossible to find an alternative enzyme that will accept the unnatural substrate enantiomer. This is not insurmountable if directed-evolution approaches are used, but it can be prohibitively expensive, especially when the desired product is in an early stage of development or required for use only as an analytical reference or standard. 

A new hydroxynitrile lyase (HNL) was isolated from the seed of Japanese apricot Prunus mume). It accepts benzaldehyde and a large number of unnatural substrates for the addition of HCN to produce the corresponding (7 )-cyanohydrins in excellent optical and chemical yields. A new high-performance liquid chromatography (HPLC)-based enantioselective assay technique was developed for the enzyme, which promotes the addition of KCN to benzaldehyde in a buffered solution (pH 4.0). Asymmetric synthesis of (7 )-cyanohydrins by a new HNL is described (Figure 8.4). ... 

Microorganisms with both the required activities were discovered that surprisingly acted with 100% specificity on the isomer of an unnatural substrate, and that did not further metabolise the lysine product. 

The Pseudomonas fluorescens KdgA was shown to accept several polar-sub-stituted aldehydes, albeit at rates much lower ( < 1%) than the phosphorylated natural substrate 12 (Table 3) [137]. Simple aliphatic or aromatic aldehydes were not converted. Synthetic utility and high stereoselectivity with unnatural substrates were demonstrated by conversion of both the D-configurated glycer-aldehyde (d-15) and lactaldehyde (d-16) to form the respective (4S)-configurated adducts 17 and 18 at the mmol scale. 

Deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS), the enzyme isolated from Cephalosporium acremonium catalysing587 the ring expansion of penicillin N, 473, to deacetoxycephalosporin (DAOC), 474, and the hydroxylation of 474 to deacetylcephalosporin C(DAC), 475, which in vivo is acetylated by a different enzyme to give cephalosporin C, 476 (equation 284), converts also the unnatural substrate exomethylene cephalosporin C, 477a, directly to DAC, 475 (equation 285). 

Figure 10.9 Unnatural substrate specificity and enantiospecificity of DERA (DeSantis, 2003).

Fig. 6 In vivo reprogramming of alkaloid biosynthesis in hairy roots of C. roseus by introduction of a mutant cDNA of the key enzyme strictosidine synthase (STR) with broader, unnatural substrate specificity leading to diversification of alkaloid content in roots following long-term feeding with 5-substituted tryptamines (X = Cl, Br, Me) [78]...

Acidic proteinoids accelerate the hydrolysis of the unnatural substrate, p-nitrophenyl acetate 7,8). P-Nitrophenyl acetate has been used as a substrate for both natural esterases and esterase models. The imidazole ring of histidine is involved in the active site of a variety of enzymes, including hydrolytic enzymes. Histidine residues of proteinoid play a key role in the hydrolysis, the contribution to activity of residues of lysine and arginine is minor, and no activity is observed for proteinoid containing no basic amino acid 7). 

Proteinoid microspheres containing zinc hydrolyze the natural substrate, adenosine triphosphate (ATP) as well as the unnatural substrates, p-nitrophenylacetate or p-nitrophenyl phosphate. The significance resides in the fact that the energy for most biosyntheses is provided by the hydrolysis of ATP. Zinc, magnesium and other metal salts are known to catalyze the hydrolysis of ATP 10). Proteinoid microspheres containing zinc as a cofactor have an activity for hydrolysis of ATP 11 12). 

As discussed above, the purification and reconstitution of active PKSs from a variety of heterologous expression systems (including E. coli) are now feasible. Given the substantial tolerance of PKSs toward altered substrates and intermediates, it should therefore be possible to exploit this catalytic potential in a far more powerful way in cell-free systems than in intracellular systems. The primary limitations are with regard to the scale of synthesis. Attempts to stabilize and reuse the enzymes, in conjunction with the development of cheaper sources of natural and unnatural substrates and recycling systems for NADPH, should go a long way toward ameliorating this limitation. 

R Pieper, S Ebert-Khosla, DE Cane, C Khosla. Erythromycin biosynthesis kinetic studies on a fully active modular polyketide synthase using natural and unnatural substrates. Biochemistry 35 2054-2060, 1996. 

Since natural enzymes are unable to accept all of the unnatural substrates that they are called upon to accept for organic synthesis applications, alternative biocatalysts with expanded substrate specificity are needed. One approach toward the generation of new biocatalysts is to exploit the molecular diversity of the immune system by recruiting catalytic antibodies as protein catalysts. 

Design of RNA molecules with novel catalytic functions called ribozymes (ribonucleotide enzymes) started out from the reprogramming of naturally occurring molecules to accept unnatural substrates [32, 33] A specific RNA cleaving ribozyme, a class I (self-splicing) intron, was modified through variation and selection until it operated efficiently on DNA. The evolutionary path of such a transformation of catalytic activity has been recorded in molecular detail [34]. The basic problem in the evolutionary design of new catalysts is the availability of appropriate analytical tools for the detec-... 

On the other hand the enantioselectivity of yeast ADH in the reduction of ketones has been unclear. When whole yeast cells are employed the reduction of methyl ketones also leads to mainly (S)-configurated alcohols, however the optical purities of these alcohols are only moderate ( ). Mac Leod et al. rationalized this lack of 100% stereoselectivity by the assumption that alcohol dehydrogenase may be the only enzyme involved in this reduction but that it is only partially stereoselective when acting on these unnatural substrates. 

Of special interest is the elaboration of conditions for large scale preparations, reactions in semi-aqueous systems, and synthetic applications of less specific enzymes for unnatural substrates. 

Although static and dynamic disorder had been detected for other enzyme substrate systems before [10-12], one could argue that its observation in this case is an artifact of the way, how the experiments were performed. The nonspecific immobilization procedure might result in static disorder. Enzymes with different orientations on the surface might possess a different lid mobility and accessibility of the active site and, as a result, show different activities. And the use of the highly unnatural substrate might be a possible source of dynamic disorder. An alternative detection scheme for this class of enzymes, which solves these shortcomings, will be presented in Sect. 25.3. 

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