Mandelate enzyme

If RCOOH is a comparatively simple organic acid and R OH a monohydric alcohol then the enzyme is called an esterase. Examples of such esters are ethyl butyrate, C3H7COOC2H5, and ethyl mandelate, CeHjCH(OH)COOC2Hj. 

M. Mandels and E.T. Reese, in Advances in Enzymic Hydrolysis of Celltdose and Related Materials, ed. E. T. Reese, Pergamon, Oxford, 1963, pp. 115-157. 

These results are compatible with an evolutionary history in which the new enzyme activity of mandelate racemase has evolved from a preexisting enzyme that catalyzes the basic chemical reaction of proton abstraction and formation of an intermediate. Subsequent mutations have modified the... 

Figure 4.9 Mechanisms of the reactions catalyzed by the enzymes mandelate racemase (a) and muconate lactonizing enzyme (b). The two overall reactions are quite different a change of configuration of a carbon atom for mandelate racemase versus ring closure for the lactonizing enzyme. However, one crucial step (red) in the two reactions is the same addition of a proton (blue) to an intermediate of the substrate (red) from a lysine residue of the enzyme (E) or. In the reverse direction, formation of an intermediate by proton abstraction from the carbon atom adjacent to the carboxylate group.

Neidhart, D.J., et al. Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous. Nature 347 ... 

Beanie, S. L., and Wolfenden, R., 1997. Mandelate racemase in pieces Effective concentrations of enzyme fnnctional groups in the transition state. Biochemistry 36 1646-1656. 

The addition of HCN to aldehydes or ketones produces cyanohydrins (a-hydroxy nitriles). Cyanohydrins racemize under basic conditions through reversible loss of FiCN as illustrated in Figure 6.30. Enantiopure a-hydroxy acids can be obtained via the DKR of racemic cyanohydrins in the presence of an enantioselective nitriletransforming enzyme [86-88]. Many nitrile hydratases are metalloenzymes sensitive to cyanide and a nitrilase is usually used in this biotransformation. The DKR of mandelonitrile has been extended to an industrial process for the manufacture of (R)-mandelic acid [89]. 

The a carbon of mandelic acid is sp hybridized. The corresponding carbons of both a-phenylglycidic acid, 49, and the carbanion intermediate 48 are neither sp hybridized nor sp hybridized, but presumably between these two extremes. It is therefore possible that the a-phenylglycidic acid is restricted to a conformation which resembles a transition state in the racemization process, a transition state which would have much of the character of the intermediate 48, and for which the enzyme would presumably have a high affinity (1). 

The basal medium of Mandels (Mandels et al., 1976) was used with the following modifications it was buffered with 3 g/1 of sodium nitrate to pH 5.5 and supplemented with 1% w/v citrus pectin " Sigma" or other carbon sources. For enzyme production, 50 ml medium in 250 ml erlemneyer flasks were inoculatedwith spores (10 spores /ml ) exept for the non sporulating Pol 6 strain, where mycelium was used. The culture were incubated at 30° C on a rotary shaker (150 rev mn -1) for 5 days. The culture broth was filtered (Millipore 0.45 pm ) and the supernatant was analysed for pectinolytic activities, reducing sugars and proteins. 

Hegeman GD (1966) Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida I synthesis of enzymes of the wild type. J Bacteriol 91 1140-1154. 

Halpin RA, GD Hegeman, GL Kenyon (1981) Carbon-13 nuclear magnetic resonance studies of mandelate metabolism in whole bacterial cells and in isolated, in vivo cross-linked enzyme complexes. Biochemistry 20 1525-1533. 

The mandelate pathway in Pseudomonas putida involves successive oxidation to benzoyl formate and benzoate, which is further metabolized via catechol and the 3-ketoadipate pathway (Figure 8.35a) (Hegeman 1966). Both enantiomers of mandelate were degraded through the activity of a mandelate racemase (Hegeman 1966), and the racemase (mdlA) is encoded in an operon that includes the next two enzymes in the pathway—5-mandel-ate dehydrogenase (mdlB) and benzoylformate decarboxylase (mdlC) (Tsou et al. 1990). 

An enantioselective nitrilase from Pseudomonas putida isolated from soil cultured with 2 mM phenylacetonitrile was purified and characterized. This enzyme is comprised of 9-10 identical subunits each of 43 kDa. It exhibits a pH optimum at 7.0 and a temperature optimum at 40 °C (Ty2 = 160 min) and requires a reducing environment for activity. This nitrilase was shown to have an unusually high tolerance for acetone as co-solvent, with >50% activity retained in the presence of 30% acetone. The kinetic profile of this nitrilase reveals KM= 13.4mM, cat/ M = 0-9s 1mM 1 for mandelonitrile, ZfM = 3.6mM, kclJKM 5.2 s him-1 for phenylacetonitrile, and KM = 5.3 mM, kC lt/KM = 2.5 s 1 him 1 for indole 3-acetonitrile. Preliminary analysis of this enzyme with 5 mM mandelonitrile revealed formation of (/t)-mandelic acid with 99.9% ee [59]. 

A patent procedure for formation of compounds 19 from simple tartaric acid derivatives has appeared <06USP047129> and various new routes to chiral dioxolanones include synthesis of dioxolan-2-ones either by transition metal-mediated asymmetric synthesis <06T1864> or enzyme-mediated kinetic resolution <06H(68)1329> and a new synthesis of the chiral dioxolan-4-ones 21 from lactic or mandelic acid involving initial formation of intermediates 20 with trimethyl orthoformate in cyclohexane followed by reaction with pivalaldehyde <06S3915>. 

Enzymes exhibit specificity as regards their behaviour towards stereo-isomerides. Pasteur s biological resolution of racemic acid with moulds depends on this fact, and such moulds may also be used for the resolution of racemic mandelic acid, for the partial digestion of racemic polypeptides according to E. Fischer s method, and in numerous other processes. 

A different concept of chiral recognition was used by Lehn et al. (1978) for the differentiation between pairs of enantiomeric anions. Following the terminology used for metallo-enzymes, the chiral crown ether [309] acts as an apo-receptor, complexing a metal cation and thus becoming a chiral metal receptor that may discriminate between enantiomeric anions (cascade-type complexation). Extraction experiments with racemic mandelic acid dissolved in... 

Mannering, G.J. Microsomal enzyme systems which catalyze drug metabolism. In La Du, B.N., Mandel, H.G. and Way, E.L. (Eds.) Fundamentals of Drug Metabolism and Disposition. Baltimore, Williams.(1971). 

To elucidate the metabolic pathway of phenylmalonic acid, the incubation broth of A. bronchisepticus on phenylmalonic acid was examined at the early stage of cultivation. After a one-day incubation period, phenylmalonic acid was recovered in 80% yield. It is worthy of note that the supposed intermediate, mandelic acid, was obtained in 1.4% yield, as shown in Eq. (8). The absolute configuration of this oxidation product was revealed to be S. After 2 days, no metabolite was recovered from the broth. It is highly probable that the intermediary mandelic acid is further oxidized via benzoylformic acid. As the isolated mandelic acid is optically active, the enzyme responsible for the oxidation of the acid is assumed to be S-specific. If this assumption is correct, the enzyme should leave the intact l -enantiomer behind when a racemic mixture of mandelic acid is subjected to the reaction. This expectation was nicely realized by adding the racemate of mandelic acid to a suspension of A. bronchisepticus after a 4-day incubation [4]. 

The present reaction was proven to occur even when the microorganism had been grown on peptone as the sole carbon source. These results lead to the conclusion that this enzyme system is produced constitutively. In the case of man-delate-pathway enzymes in Pseudomonas putida, (S)-mandelate dehydrogenase was shown to be produced in the presence of an inducer (mandelic acid or benzoylformic acid) [5]. Thus, the expression of the present oxidizing enzyme of A. bronchisepticus is different from that of R putida. 

Wheat bran. Profuse growth of T, reesei QMY-1 was observed within first 5 or 3 days of SSF on wheat bran without any additional nutrients or with nutrients from the Mandels and Weber medium (22), respectively. In spite of good growth on wheat bran without additionsil nutrients, very low activities of all the enzymes were obtained (Table IV). On wheat bran with nutrients, the activities of FP cellulase, -glucosidase, and xylanase increased to 8.3, 5.1, and 13875 times, respectively, but the activities of all the enzymes still remained very low compared to those obtained on wheat straw in SSF or LSF (Tables I and IV). 

Other Proteins The ouabain-binding site on (Na /K -adenosine-5 -triphosphatase, 46, 523 penicillin isocyanates for /3-lactamase, 46, 531 active site-directed addition of a small group to an enzyme the ethylation of ludferin, 46, 537 mandelate racemase, 46, 541 d imethylpyrazole carboxamidine and related derivatives, 46, 548 labeling of catechol O-methyltransferase with N-haloace-tyl derivatives, 46, 554 affinity labeling of binding sites in proteins by sensitized photooxidation, 46, 561 bromocolchicine as a iabei for tubuiin, 46, 567. 

Mandelate racemase, another pertinent example, catalyzes the kinetically and thermodynamically unfavorable a-carbon proton abstraction. Bearne and Wolfenden measured deuterium incorporation rates into the a-posi-tion of mandelate and the rate of (i )-mandelate racemi-zation upon incubation at elevated temperatures. From an Arrhenius plot, they obtained a for racemization and deuterium exchange rate was estimated to be around 35 kcal/mol at 25°C under neutral conditions. The magnitude of the latter indicated mandelate racemase achieves the remarkable rate enhancement of 1.7 X 10, and a level of transition state affinity (K x = 2 X 10 M). These investigators also estimated the effective concentrations of the catalytic side chains in the native protein for Lys-166, the effective concentration was 622 M for His-297, they obtained a value 3 X 10 M and for Glu-317, the value was 3 X 10 M. The authors state that their observations are consistent with the idea that general acid-general base catalysis is efficient mode of catalysis when enzyme s structure is optimally complementary with their substrates in the transition-state. See Reference Reaction Catalytic Enhancement... 

This iron-dependent enzyme [EC 1.14.16.6], also known as L-mandelate 4-hydroxylase, catalyzes the reaction of (5)-2-hydroxy-2-phenylacetate with tetrahydropteridine and dioxygen to produce (5)-4-hydroxymandelate, dihy-dropteridine, and water. 

BFD from Pseudomonas putida has been characterized in detail with respect to its biochemical properties [4, 5] and 3D structure [6, 7]. Like other enzymes of this class, BFD is a homotetramer with a subunit size of about 56 kDa. The four active sites are formed at the interfaces of two subunits. The structure was published in 2003 [7] and contains the competitive inhibitor (R)-mandelate bound to the active sites, allowing model-based predictions about the interactions between active site residues and the substrate. 

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