Phenylalanine dehydrogenase PheDH

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. ... 

Table 7. Substrate Specificity of Phenylalanine Dehydrogenase (PheDH) from Bacillus sphaericus SCRC-R79a10...

Optically pure opine-tjrpe secondary amine carboxylic acids were also synthesized from amino acids and their analogs, such as L-methionine, L-isoleucine, L-leucine, L-valine, L-phenylalanine, L-alanine, L-threonine, L-serine, and L-phenylalaninol, and a-keto acids, such as glyoxylic, pyruvic, and 2-oxobutyric acids, using the enzyme with regeneration of NADH with FDH from Moraxella sp. C-1 [13]. The absolute configuration of the nascent asymmetric center of the opines was of the D stereochemistry with > 99.9% e.e. One-pot synthesis of N-[l-D-(carboxyl)ethyl]-L-phenylalanine from phenylpyruvic and pyruvic acid by using ODH, FDH, and phenylalanine dehydrogenase (PheDH) from Bacillus sphaericus... 

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. 

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. 

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). 

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

FDH, Formate dehydrogenase L-HicDH, L-Hydroxyisocaproate dehydrogenase LeuDH, Leucine dehydrogenase PheDH, Phenylalanine dehydrogenase. 

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. 

At the beginning of the 1980s, the wide screening of aromatic amino acid dehydrogenases led to the discovery of PheDH in Brevibacterium species [26]. The enzyme was isolated from several mesophiles—Bacillus sphaericus [27], Sporosarcina ureae [27], B. badius [28], Rhodococcus sp. [29], Nocardia sp. [30] and Microbacterium sp. [31], and also from the thermophile T. intermedius [32]—and characterize (Table 5). The enzyme acts on L-norleucine, L-methionine, L-norvaline, and L-tyrosine besides L-phenylalanine in the presence of NAD, although slowly. L-Tryptophan, L-alanine, and D-phenyManine are inert as the substrate. The enzyme shows lower substrate specificity for 2-oxo acids than that for amino acids like AlaDH and LeuDH. The values for ammonia are more than 70 mM. The T. intermedius PheDH is the most thermostable and a useful catalyst for industrial and clinical applications. The enzyme is easily and effectively purified from the recombinant E. coli [6] and commercially available (Unitika Ltd.). 

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