L-Phenylalanine dehydrogenase

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. 

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. ... 

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). 

Reductive amination reactions of keto acids are performed with amino acid dehydrogenases. NAD-dependent leucine dehydrogenase from Bacillus sp. is of interest for the synthesis of (S)-fert.-leucine [15-17]]. This chiral compound has found widespread application in asymmetric synthesis and as a building block of biologically active substances. The enzyme can also be used for the chemoenzy-matic preparation of (S)-hydroxy-valine [18] and unnatural hydrophobic bran-ched-chain (S)-amino acids. NAD-dependent L-phenylalanine dehydrogenase from Rhodococcus sp. [19] has been used for the synthesis of L-homophenyl-alanine ((S)-2-Amino-4-phenylbutanoic acid) [9]. These processes with water-soluble substrates and products demonstrate that the use of coenzymes must not... 

In one of the first examples, the reductive amination of phenylpyruvate catalyzed by a NADH-dependent L-phenylalanine dehydrogenase (PheDH) was coupled with the in situ generation of the substrate from acetamidocinnamic add (ACA) by a suitable acylase, thus avoiding both substrate inhibition and instability (Scheme 11.12a) [20]. An intracellular acylase was selected from a Brevibacterium strain and employed in this one-pot process at ACA concentrations up to 0.3 M with quantitative conversions into the desired product L-phenylalanine. 

The aim of this complex system is to obtain optically pure amino acid from the corresponding racemate without the necessity to separate the a-keto acid. D-Methionine is oxidized to 2-oxo-4-methylthiobutyric acid by D-amino acid oxidase (D-AAO). Catalase is used to decompose formed hydrogen peroxide. Reduction of 2-oxo-4-methylthiobutyric acid to L-methionine is accompHshed with L-phenylalanine dehydrogenase (L-PheDH), requiring a coenzyme NADH. The latter needs regeneration, which explains a necessity to include formate dehydrogenase in this system. 

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). 

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. 

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... 

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. 

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]. 

J. J. Venit, L. J. Szarka, and R. N. Patel, Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius, Enzyme Microb. Technol. 2000b, 26, 348-358. 

Fig. 39 Conversion of 5-(l,3-dioxolan-2-yl)-2-oxo-pentanoid acid to allysine ethylene acetal by reductive amination using phenylalanine dehydrogenase (PDH) and formate dehydrogenase (FDH) for cofactor recycling...

L-Phenylalanine,which is derived via the shikimic acid pathway,is an important precursor for aromatic aroma components. This amino acid can be transformed into phe-nylpyruvate by transamination and by subsequent decarboxylation to 2-phenylacetyl-CoA in an analogous reaction as discussed for leucine and valine. 2-Phenylacetyl-CoA is converted into esters of a variety of alcohols or reduced to 2-phenylethanol and transformed into 2-phenyl-ethyl esters. The end products of phenylalanine catabolism are fumaric acid and acetoacetate which are further metabolized by the TCA-cycle. Phenylalanine ammonia lyase converts the amino acid into cinnamic acid, the key intermediate of phenylpropanoid metabolism. By a series of enzymes (cinnamate-4-hydroxylase, p-coumarate 3-hydroxylase, catechol O-methyltransferase and ferulate 5-hydroxylase) cinnamic acid is transformed into p-couma-ric-, caffeic-, ferulic-, 5-hydroxyferulic- and sinapic acids,which act as precursors for flavor components and are important intermediates in the biosynthesis of fla-vonoides, lignins, etc. Reduction of cinnamic acids to aldehydes and alcohols by cinnamoyl-CoA NADPH-oxido-reductase and cinnamoyl-alcohol-dehydrogenase form important flavor compounds such as cinnamic aldehyde, cin-namyl alcohol and esters. Further reduction of cinnamyl alcohols lead to propenyl- and allylphenols such as... 

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