Enzymes substrate tolerance

Both reactions are mechanistically related and take place in a translanti-maaner [1572, 1573], with protonation occurring from the re-side (Scheme 2.210). These close mechanistic similarities may be explained by the fact that some of these enzymes show a remarkable degree of amino acid homology [1574]. Within this group of enzymes, substrate tolerance is rather narrow, but the stereoselectivities observed are exceptionally high. 

Styrene was successfully oxidized to the S-product both by xylene monooxygenase from P. putida mt-2 [113] and styrene monooxygenase from Pseudomonas sp.VLB120 [114] (Scheme 9.13), with the latter enzyme displaying a particularly large substrate tolerance with excellent stereoselectivity (>99% ee). In this context it is interesting to note that both xylene monooxygenase as well as chloroperoxidase are very selective for mono-epoxidation in case of presence of multiple alkene functionalities [115]. 

Catalytic C—C coupling is particularly valuable in asymmetric synthesis because of its potential for stereodivergent product generation [13], by which multiple stereoisomeric products can be derived from common synthetic building blocks (Eigure 10.1). Obviously, such a synthetic strategy depends on the prevalence of related stereocomplementary enzymes that must have a similarly broad substrate tolerance. 

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

A more general access to biologically important and structurally more diverse aldose isomers makes use of ketol isomerases for the enzymatic interconversion of ketoses to aldoses. For a full realization of the concept of enzymatic stereodivergent carbohydrate synthesis, the stereochemically complementary i-rhamnose (Rhal EC 5.3.1.14) and i-fucose isomerases (Fuel EC 5.3.1.3) from E. coli have been shown to display a relaxed substrate tolerance [16,99,113,131]. Both enzymes convert sugars and their derivatives that have a common (3 J )-OH configuration, but may deviate in... 

A biochemically related benzaldehyde lyase (BAL) (EC 4.1.2.38) catalyzes the same carboligation reactions, but with opposite (J )-selectivity (mf-110) [178]. All these enzymes seem to display a rather useful substrate tolerance for variously substituted aldehyde precursors. 

Whereas SHMT in vivo has a biosynthetic function, threonine aldolase catalyzes the degradation of threonine both l- and D-spedfic ThrA enzymes are known [16,192]. Typically, ThrA enzymes show complete enantiopreference for their natural a-D- or a-t-amino configuration but, with few exceptions, have only low specificity for the relative threo/erythro-configuration (e.g. (122)/(123)) [193]. Likewise, SHMT is highly selective for the L-configuration, but has poor threo/erythro selectivity [194]. For biocatalytic applications, the knovm SHMT, d- and t-ThrA show broad substrate tolerance for various acceptor aldehydes, notably induding aromatic aldehydes [193-196] however, a,P-unsaturated aldehydes are not accepted [197]. For preparative reactions, excess of (120) must compensate for the unfavorable equilibrium constant [34] to achieve economical yields. 

Figure 3.2. The chemical diversity that is characteristic of NPs can be considered to arise in two phases. In the first phase, a few precursors are joined together in a few similar ways (using a modular or iterative processes) to produce families of structures that provide the basic carbon skeletons that characterise the group. In the second phase, enzymes with broad substrate tolerances tailor the skeletons in versatile ways to generate even greater diversity.

Evidence to support this proposition that enzymes involved in NP metabolism would possess broad substrate tolerance... 

The ready availability of the transketolase (TK E.C. 2.2.1.1) from E. coli within the research collaboration in G. A. Sprenger s group suggested the joint development of an improved synthesis of D-xylulose 5-phosphate 19, which was expensive but required routinely for activity measurements [27]. In vivo, transketolase catalyzes the stereospecific transfer of a hydroxyacetyl nucleophile between various sugar phosphates in the presence of a thiamine diphosphate cofactor and divalent cations, and the C2 donor component 19 offers superior kinetic constants. For synthetic purposes, the enzyme is generally attractive for its high asymmetric induction at the newly formed chiral center and high kinetic enantioselectivity for 2-hydroxyaldehydes, as well as its broad substrate tolerance for aldehyde acceptors [28]. 

C-C bond formation renders the addition essentially irreversible. Our first preparative screening efforts have shown that the enzyme exhibits a broad substrate tolerance, particularly by accepting variously C5-modified NeuSAc derivatives as substrates with complete stereocontrol [20, 21]. Thus, the enzyme seems to be practically equivalent to the commercial aldolase, except that reactions attain complete conversion without a need to drive an equilibrium by a large excess of substrate, which strongly simplifies product isolation. 

Controversy remains in the determination of substrate tolerance for KdgA/KhgA aldolases from different sources. Early assay studies with KhgA prepared [127-129] from rat liver concluded that the catalyst had an unusually wide ranging tolerance for nucleophilic components, including a number of 3-substituted pyruvate derivatives as well as pyruvaldehyde, acetaldehyde, and pyruvic esters [135], Later, other workers using enzymes from rat or bovine liver and from E. coli reported their inability to reproduce these results but noted a rather limiting specificity [136]. 

Like a number of other aldolases the FucA enzyme is now also offered commercially. Overall practical features make the FucA quite similar to the RhuA enzyme, as is illustrated by its high stability in the presence of Zn2 + ions, by its broad substrate tolerance for variously substituted aldehydes at useful reaction rates (Table 5), and by a high asymmetric induction for (3R,4R)-cis stereoselectivity by si-face addition to the aldehyde carbonyl [195,355]. Al-... 

The degradation of nicotinic acid by Clostridium barkeri involves the cleavage of the intermediate 2,3-dimethylmalate 132 from which propionic and pyruvic acids are formed by a specific lyase (EC 4.1.3.32). In the reverse direction, the enzyme must have the unusual capacity to deprotonate propionic acid at the a-carbon instead of the carboxylic acid function, or next to an anionic car-boxylate. Purified dimethylmalic acid aldolase has been used to catalyze the stereospecific addition of 133 to the oxoacid acceptor, yielding the (2R,3S) configurated dimethylmalic acid 132 at the multi-gram scale [381]. The substrate tolerance of this enzyme has not yet been determined. 

A corresponding ThrA has been detected in a number of strictly anaerobic bacteria, and the enzyme from Clostridium pasteurianum has been purified and shown to be highly selective for L-threonine 150 [457]. A corresponding L-specific catalyst has also been purified and crystallized from cells of the yeast Candida humicola. Very recently, the latter enzyme was reinvestigated for synthetic purposes and found to have a very broad substrate tolerance for the aldehyde acceptor, notably including variously substituted aliphatic and aro-... 

A ThrA enzyme that is highly specific for L-threo stereoisomers has been detected in Streptomyces amakusaensis. The purified enzyme has been employed for the efficient resolution of chemically generated racemic f/ireo-phenylserines 156, substituted at the p-position (H, OH, Br, N02), to furnish enantiomerically pure D-amino acids 157 as the (2R,3S) diastereomers [459], Preliminary screening results suggest a broad substrate tolerance [460]. 

The Sorghum (S)-oxynitrilase exclusively catalyzes the addition of hydrocyanic acid to aromatic aldehydes with high enantioselectivity, but not to aliphatic aldehydes or ketones [519, 526], In contrast, the Hevea (S)-oxynitrilase was also found to convert aliphatic and a,/ -unsaturated substrates with medium to high selectivity [509, 527]. The stereocomplementary almond (R)-oxynitrilase likewise has a very broad substrate tolerance and accepts both aromatic, aliphatic, and a,/ -unsaturated aldehydes [520, 521, 523, 528, 529] as well as methyl ketones [530] with high enantiomeric excess (Table 9). It is interesting to note that this enzyme will also tolerate sterically hindered substrates such as pivalaldehyde and suitable derivatives 164 which are effective precursors for (R)-pantolactone 165 [531],... 

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