Dehydrogenases phenylalanine dehydrogenase

OCTANOL DEHYDROGENASE OCTOPINE DEHYDROGENASE PHENYLALANINE DEHYDROGENASE POLY(ADP-RIBOSE) SYNTHETASE (or, SYNTHASE)... 

Two reactions for the production of L-phenylalanine that can be performed particularly well in an enzyme membrane reactor (EMR) are shown in reaction 5 and 6. The recently discovered enzyme phenylalanine dehydrogenase plays an important role. As can be seen, the reactions are coenzyme dependent and the production of L-phenylalanine is by reductive animation of phenylpyruvic add. Electrons can be transported from formic add to phenylpyruvic add so that two substrates have to be used formic add and an a-keto add phenylpyruvic add (reaction 5). Also electrons can be transported from an a-hydroxy add to form phenylpyruvic add which can be aminated so that only one substrate has to be used a-hydroxy acid phenyllactic acid (reaction 6). 

Figure 1. Schematic outline of various products and associated enzymes from the shikimate and phenolic pathways in plants (and some microorganisms). Enzymes (1) 3-deoxy-2-oxo-D-arabino-heptulosate-7-phosphate synthase (2) 5-dehydroquinate synthase (3) shikimate dehydrogenase (4) shikimate kinase (5) 5-enol-pyruvylshikimate-3-phosphate synthase (6) chorismate synthase (7) chorismate mutase (8) prephenate dehydrogenase (9) tyrosine aminotransferase (10) prephenate dehydratase (11) phenylalanine aminotransferase (12) anthranilate synthase (13) tryptophan synthase (14) phenylalanine ammonia-lyase (15) tyrosine ammonia-lyase and (16) polyphenol oxidase. (From ACS Symposium Series No. 181, 1982) (62).

A typical example of these analytical systems is a manifold using bacterial luciferase for L-phenylalanine assay [228] developed with two separate nylon coils, as shown in Figure 3. The first one contained the specific L-phenylalanine dehydrogenase (L-PheDH) enzyme. 

ENZ enzyme assays, SC structural composition, MM molecular methods, IL isotopic labeling, IF isotopic fractionation, INH inhibition studies, UNK unknown, LOX lipoxogenase, EPSP synthase 5-enolpyruvylshikimate-3-phosphate, SDH shikimate dehydrogenase, PAL phenylalanine ammonium lyase, PKS polyketide synthase, NRPS nonribosomal peptide synthase 1 Gerwick 1999 2 Liu et al. 1994 3 Boonprab et al. 2003 4 Cvejic and Rohmer 1999 5 Disch et al. 1998 6 Chikaraishi et al. 2006 7 Schwender et al. 2001 8 Schwender et al. 1997 9 Mayes et al. 1993 10 Shick et al. 1999 11 Richards et al. 2006 12 Bouarab et al. 2004 13 Pelletreau et al., unpublished data 14 Dittman and Weigand 2006 15 Rein and Barrone 1999 Empty columns imply no direct evidence of these enzymes from these systems... 

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

Some enzymes with improved single amino acid specificity are commercially available. An example is phenylalanine dehydrogenase (EC 1.4.1.1), derived from bacterial sources, which acts on phenylalanine with the simultaneous conversion of NAD to NADH. Quantitation of the phenylalanine is based on determining the amount of NADH produced using standard procedures. In the direct methods, the absorbance at 340 nm is measured, whereas in the colorimetric methods, the reaction is coupled to an electron acceptor... 

A biosensor was designed where a dehydrogenase and an enlarged coenzyme are confined behind an ultrafiltration membrane. The amino acid is determined indirectly, by measuring the fluorescence of the reduced coenzyme (kex 360 nm, kfl 460 nm) produced in reaction 22, with the aid of an optical fiber. The coenzyme is regenerated with pyruvate in a subsequent step, as shown in reaction 23. This biosensor was proposed for determination of L-alanine and L-phenylalanine for monitoring of various metabolic diseases and for dietary management363. 

Synthesis of Substituted Derivatives of L-Phenylalanine and of other Non-natural L-Amino Acids Using Engineered Mutants of Phenylalanine Dehydrogenase... 

We have used a series of biocatalysts produced by site-directed mutations at the active site of L-phenylalanine dehydrogenase (PheDH) of Bacillus sphaericus, which expand the substrate specificity range beyond that of the wild-type enzyme, to catalyse oxidoreduc-tions involving various non-natural L-amino acids. These may be produced by enantiose-lective enzyme-catalysed reductive amination of the corresponding 2-oxoacid. Since the reaction is reversible, these biocatalysts may also be used to effect a kinetic resolution of a D,L racemic mixture. ... 

Seah, S.Y.K., Britton, K.L., Rice, D.W., Asano, Y. and Engel, P.C., Single amino acid substitution in Bacillus sphacricus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates. Biochemistry, 2002, 41, 11390. 

Busca, P., Paradisi, E., Moynihan, E., Maguire, A.R. and Engel, P.C., Enantioselective synthesis of non-natural amino acids using phenylalanine dehydrogenases modified by site-directed mutagenesis. Org. Biomol. Chem., 2004, 2, 2684. 

L-Phenylalanine dehydrogenase in chiral L-amino acid synthesis 76... 

In a similar exercise with D-methionine, Findrik and Vasic-Racki used the D-AAO of Arthrobacter, and for the second-step conversion of oxoacid into L-amino acid, used L-phenylalanine dehydrogenase (L-PheDH), which has a sufficiently broad specificity to accept L-methionine and its corresponding oxoacid as substrates. Efficient quantitative conversion in this latter reaction requires recycling of the cofactor NAD into NADH, and for this the commercially available formate dehydrogenase (FDH) was used (Scheme 2). 

Figure 2.16. Pathways for the synthesis and metabolism of the catecholamines. A=phenylalanine hydroxylase+pteridine cofactor+Oj B tyrosine hydroxylase+ tetrahydropteridme+Fe+ +Oj C=dopa decarboxylase+pyridoxal phosphate D= dopamine beta-oxidase+ascorbate phosphate+Cu+ +Oj E=phenylethanolamine N-methyltransferase+S-adenosylmethionine l=monoamine oxidase and aldehyde dehydrogenase 2=catechol-0-methyltransferase+S-adenosylmethionine.

R)-2-METHYLMALATE DEHYDRATASE NICOTINATE DEHYDROGENASE NITRATE REDUCTASE NITRITE REDUCTASE PHENYLALANINE MONOOXYGENASE PROLYL 3-HYDROXYLASE PROLYL 4-HYDROXYLASE PROTOCATECHUATE 3,4-DIOXYGENASE PROTOCATECHUATE 4,5-DIOXYGENASE RIESKE IRON-SULFUR PROTEIN RUBREDOXIN... 

PHENYLALANINE AMINOTRANSFERASE PHENYLALANINE AMMONIA-LYASE PHENYLALANINE DECARBOXYLASE PHENYLALANINE DEHYDROGENASE PHENYLALANINE MONOOXYGENASE PHENYLALANINE RACEMASE PHENYLALANINE AMINOTRANSFERASE AROMATIC AMINO ACID AMINOTRANSFERASE... 

PHENYLALANINE AMMONIA-LYASE DEHYDROALANINE BOROHYDRIDE REDUCTION PHENYLALANINE DECARBOXYLASE PHENYLALANINE DEHYDROGENASE Phenylalanine(histidine) aminotransferase, PHENYLALANINE AMINOTRANSFERASE PHENYLALANINE HYDROXYLASE (Phenylalanine Monooxygenase)... 

PHENYLALANINE DEHYDROGENASE PREPHENATE DEHYDRATASE PHENYLPYRUVATE SYNTHASE PHI (< or4))... 

Bacillus sphaericus phenylalanine dehydrogenase mutant (on celite)... 

The vast majority of amino acid dehydrogenases use ammonium ions as the amine donor. However, recently a novel N-methyl-L-amino acid dehydrogenase (NMAADH), from Pseudomonas putida, was isolated and used to synthesize N-methyl-L-phenylalanine 36 from phenylpyruvic acid 31 and methylamine 35 in 98% yield and greater than 99%e.e. (Scheme 2.15). The enzyme was shown to accept a number of different ketoacids and also use various amine donors. Glucose dehydrogenase from Bacillus suhtilis was used to recycle the NADPH cofactor [17]. 

HicDH = Hydroxycaproate dehydrogenase PheDH = Phenylalanine dehydrogenase Scheme 6.14 Enzymatic conversion of an a-hydroxy acid to an a-amino acid. 

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