S hydrolase

The four-step synthesis of a, -diaminocaprolactam shown in Figure 5.29 is part ofa chemoenzymatic route to (S)-lysine, an essential amino add in our diet [135], The racemic caprolactam (azepan-2-one) product is then hydrolyzed selectively to (S)-lysine, using an immobilized (S)-hydrolase enzyme. 

Scherer T.M., Clinton Fuller R., Lenz R.W., Goodwin S. Hydrolase activity of an extracellular depolymerase from Aspergillus famigatus with bacterial and synthethic polyesters, Polym. Deg. Stab. 64(1999)267. 

The metabolism of foreign compounds (xenobiotics) often takes place in two consecutive reactions, classically referred to as phases one and two. Phase I is a functionalization of the lipophilic compound that can be used to attach a conjugate in Phase II. The conjugated product is usually sufficiently water-soluble to be excretable into the urine. The most important biotransformations of Phase I are aromatic and aliphatic hydroxylations catalyzed by cytochromes P450. Other Phase I enzymes are for example epoxide hydrolases or carboxylesterases. Typical Phase II enzymes are UDP-glucuronosyltrans-ferases, sulfotransferases, N-acetyltransferases and methyltransferases e.g. thiopurin S-methyltransferase. 

In all the reported examples, the enzyme selectivity was affected by the solvent used, but the stereochemical preference remained the same. However, in some specific cases it was found that it was also possible to invert the hydrolases enantioselectivity. The first report was again from iQibanov s group, which described the transesterification of the model compound (13) with n-propanol. As shown in Table 1.6, the enantiopreference of an Aspergillus oryzae protease shifted from the (l)- to the (D)-enantiomer by moving from acetonitrile to CCI4 [30]. Similar observations on the inversion of enantioselectivity by switching from one solvent to another were later reported by other authors [31]. 

The lipase (PAL) used in these studies is a hydrolase having the usual catalytic triad composed of aspartate, histidine, and serine [42] (Figure 2.6). Stereoselectivity is determined in the first step, which involves the formation of the oxyanion. Unfortunately, X-ray structural characterization of the (S)- and (J )-selective mutants are not available. However, consideration of the crystal structure of the WT lipase [42] is in itself illuminating. Surprisingly, it turned out that many of the mutants have amino acid exchanges remote from the active site [8,22,40]. 

Several reports regarding the directed evolution of enantioselective epoxide hydrolases (EHs) have appeared [23,57-59]. These enzymes constitute important catalysts in synthetic organic chemistry [4,60]. The first two reported studies concern the Aspergillus niger epoxide hydrolase (ANEH) [57,58]. Initial attempts were made to enhance the enantioselectivity of the AN E H -catalyzed hydrolytic kinetic resolution of glycidyl phenyl ether (rac-19). The WT leads to an Evalue of only 4.6 in favor of (S)-20 (see Scheme 2.4) [58]. 

Figure 23-7. Conversion of arachidonicacid to leukotrienesand lipoxins of series 4 via the lipoxygenase pathway. Some similar conversions occur in series 3 and 5 leukotrienes. (HPETE, hydroperoxyeicosatetraenoate HETE, hydroxyeicosatetraenoate , peroxidase (2), leukotriene A4 epoxide hydrolase , glutathione S-transferase ...

Figure 53-1. Simplified scheme showing how metabolism of a xenobiotic can result in cell injury, immunologic damage, or cancer. In this instance, the conversion of the xenobiotic to a reactive metabolite is catalyzed by a cytochrome P450,and the conversion of the reactive metabolite (eg, an epoxide) to a nontoxic metabolite is catalyzed either by a GSH S-transferase or by epoxide hydrolase.

BHANDARI s D, GREGORY J F 3rd (1990) Inhibition by selected food components of human and porcine intestinal pteroylpolyglutamate hydrolase activity. ,4m J Clin Nutr. 51 87-94. 

The crystal structure of the HNL isolated from S. bicolor (SbHNL) was determined in a complex with the inhibitor benzoic acid." The folding pattern of SbHNL is similar to that of wheat serine carboxypeptidase (CP-WII)" and alcohol dehydrogenase." A unique two-amino acid deletion in SbHNL, however, is forcing the putative active site residues away from the hydrolase binding site toward a small hydrophobic cleft, thereby defining a completely different active site architecture where the triad of a carboxypeptidase is missing. 

Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases Phenolic polymers, polyanilines, vinyl polymers Polysaccharides, cyclic oligosaccharides, polyesters Polysaccharides, polyesters, polycarbonates, poly(amino acid)s, polyphosphates... 

The discovery of these enzymes enables a better structural characterisation of the hairy (ramified) regions of pectin, as already demonstrated by Schols et al. (1990b) and also of native plant cell wall pectin (Schols et al., 1995). In this study we show how the two exo-enzymes of the above described series, the RG-rhamnohydrolase and the RG-galacturonohydrolase, can be used as tools in the characterisation of unknown RG fragments. These unknown fragments were the products of RG-hydrolase or RG-lyase action toward linear RG oligomers (RGO s), which were produced by acid hydrolysis of sugar beet pulp. 

AH substrates (varying between 0.018 and 0.05% w/v) were incubated in 50 mM sodium acetate buffer pH 5.0, containing 0.01% w/v sodium azide, at 40 °C for 24 h. RGO s were treated with 2.6 pg RG-gaiacturonohydrolase per mg substrate. When RGO s were sequentially treated with the exo-enzymes to form smaller oligomers, the RG-galacturonohydrolase and the RG-rhamnohydrolase were used in amounts between 2.4 and 2.8 pg and between 9 and 18 pg per mg substrate respectively. RGO s were incubated with 0.18 pg RG-hydrolase and with 0.42 pg RG-lyase per mg substrate. Subsequent incubation of the RG-hydrolase/RG-lyase digest with the exo-enzymes was carried out with 6 pg of RG-galacturonohydrolase and with 16 pg RG-rhamnohydrolase per mg substrate. 

The results confirm that RG-hydrolase is a true rhamnogalacturonase, as it splits an alternating RG chain, and furthermore that it cleaves between GalA and Rha in the main chain by a mechanism of hydrolysis (Schols et al., 1990a). Similarly, the products from the RGO s with higher DP s were characterised. 

Figure 2 Locations of cleavage of RGO s by RG-hydrolase. Differently dotted arrows indicate different cleavage options. A short arrow indicates a secondary cleavage, see text. Explanation of symbols, see table 1. Numbers refer to degree of polymerisation.

The authors wish to thank Maijo Searle-van Leeuwen for purification of the P-galactosidase from Aspergillus niger and Novo Nordisk Ferment Ltd Dittingen (Switzerland) for assistance in the purification of the RG-hydrolase and Novo Nordisk A/S Bagsvaerd (Denmark) for the gift of the crude recombinant RG-lyase. 

Fischer F, S Kunne, S Fetzner (1999) Bacterial 2,4-dioxygenases new members of the a/p hydrolase-fold superfamily of enzymes functionally related to serine hydrolases. J Bacterial 181 5725-5733. 

Kataoka M, K Honda, S Shimizu (2000) 3,4-Dihydrocoumarin hydrolase with haloperoxidase activity from Acinetobacter calcoaceticus. Eur J Biochem 267 3-10. 

Picard M, J Gross, E Liibbert, S Tolzer, S Krauss, K-H van Pee (1997) Metal-free haloperoxidases as unusual hydrolases activation of HjOj by the formation of peracetic acid. Angew Chem Int Ed 36 1196-1199. 

Hareland WA, RL Crawford, PJ Chapman, S Dagley (1975) Metabolic function and properties of 4-hydroxy-phenylacetic acid 1-hydrolase from Pseudomonas acidovorans. J Bacteriol 121 272-285. 

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