PheDH substrate specificity

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. ... 

Apart from L-phenylalanine, the homolog L-homophenylalanine (L-Hph), important as a component in ACE inhibitors, can be obtained from 2-keto-4-phenyl-butyrate with PheDH[54i. The substrate specificity of PheDH from Bacillus sphaericus has been investigated by Asano et al.[551. Table 15.3-5 compares the activities of two PheDH from Rhodococcus rhodocrous[8 and Bacillus sphaericus1551 for the transformation of aromatic and aliphatic keto acids. 

The most comprehensive investigation of substrate specificity of LeuDH and PheDH has been conducted by Krix et al. (1997)[301. Table 15.3-6 lists the relative rates of various substrates. 

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

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.). 

Figure 6 Scheme of the chimeric enzyme consisting of an amino terminal domain of PheDH and a carboxy terminal domain of LeuDH. Comparison of substrate specificity of PheDH ( ), chimeric enzyme (h), and LeuDH ( ) on both amination and deamination [70]. 

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). 

PheDH from Thermoactinomyces intermedius ATCC 33 205 was utilized recently to synthesize allysine ethylene acetal [(S)-2-amino-5-(l,3-dioxolan-2-yl)-pentanoic acid (2)] from the corresponding keto acid with regeneration of NAD+ cofactor by FDH/ formate[58 (Fig. 15.3-4) the specific activity towards the keto acid was 16% compared to the standard substrate phenylpyruvate. 

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