D-amino acid dehydrogenase

ACYL-CoA DEHYDROGENASES ADENYLYLSULEATE REDUCTASE ALCOHOL OXIDASE AMINE OXIDASES d-AMINO acid DEHYDROGENASE l-AMINO acid DEHYDROGENASE d-AMINO ACID OXIDASE l-AMINO ACID OXIDASE d-ASPARTATE OXIDASE BENZOATE 1,2-DIOXYGENASE... 

In the fine chemicals industry, enantiomerically pure amino acids are mainly produced by the aminoacylase process, the amidase process, and the hydantoinase/ carbamoylase process, all three of which are suitable for I- and D-amino acids. Dehydrogenases and transaminases are now becoming established for reduction processes. 

L-amino acid amino transferase from Sinorhizobium mdUoti ATCC 51124. The enantiopure L-a-amino acid was obtained in high optical yield via the tandem action of D-amino acid dehydrogenase from the E. coli host cell (induced by L-Ala in the medium) and the cloned L-amino acid amino transferase from S. mdUoti... 

L-Lysine- -dehydrogenase (EC 1.4.1.15), an enzyme specific for the 4-pro-R hydrogen of NADPH (270), has been incubated with (2S,6R)- and (2S, 6S)-[6- H,]lysines 257a, Hc- = H, and 257a, H = H, respectively, and shown to be converted to A -piperideine-6-carboxyIate 265 with loss of the 6-pro-R hydrogen (271). The stereochemistry is very different from that exhibited by the e-aminotransferase, but it is in keeping with that of a D-amino acid dehydrogenase. 

Glutamate dehydrogenase D-amino acid oxidase Standard solution D-alanine... 

In an original application, Yasuda et al have used both l-AAO and d-AAO, and L-lysine oxidase to oxidize o ,Ci -diamino acids. The reactions produce the expected a-keto w-amino acid products, but these then spontaneously cyclize to form cyclic a-imino acids. These compounds are then substrates for the authors recently discovered A methyl amino acid dehydrogenase (NMAADH) from Pseudomonas putida, producing the pure L-cyclic amino acid (Scheme 5). 

Figure 8.6 The three dehydrogenase (oxidase) reactions in amino acid degradation. The enzymes are D-amino acid oxidase, glutamate dehydrogenase and proline oxidase (dehydrogenase). Biochemical details are given in Appendix 8.4.

Amino acid decarboxylase 744 Amino acid dehydrogenase 766 Amino acid oxidases 782 D-Amino acid oxidase 478, 790, 791s of kidney 781... 

L-6-Hydroxynorleucine, a different key chiral intermediate used for synthesis of the vasopeptidase inhibitor Omapatrilat (Vanlev ), was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beefliver (Hanson, 1999) (Figure 13.22). In an alternative process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91-97% and optical purities of > 99%. 

Many enzymes, however, do not show temperature compensation in their activities (Eckberg, 1962 Precht, 1964 Vellas, 1965 Hebb etal., 1969 Hazel and Prosser, 1970 Shkorbatov et al., 1972 Hochachka and Somero, 1973 Penny and Goldspink, 1981). Among these are catalase, peroxidase, acid phosphatase, D-amino acid oxidase, choline acetyltransferase, acetylcholinesterase and glucose-6-phosphate dehydrogenase. What is more, the same enzyme can show opposing temperature dependencies when examined in different organs and tissues of fish (Romanenko etal., 1991). 

Figure 2 Chemical conversion of 5-(4-hydroxybutyl)hydantoin (3) to racemic 6-hydroxynorleucine. Enzymatic conversion of racemic 6-hydroxynorleucine to L-6-hydroxy-norleucine (1) by D-amino acid oxidase and glutamate dehydrogenase.

For several reasons a-amino acids are ideal substrates for deracemization methods. They racemize easily by base catalysis under a number of conditions and they are racemized in Nature by the intervention of specific amino acid racemases. They are also recognized as substrates by oxidative enzymes to give the corresponding oxo-acids, in turn substrates for amino transferases and amino acid dehydrogenases. Several industrial preparations of L- and D-amino acids are based on processes of deracemization [26] or of separate two-steps resolution-racemization [27]. 

Deracemization by Stereoinversion via the Two-enzyme System D-Amino Acid Oxidase and t-Leudne Dehydrogenase... 

In a related approach, D,L-methionine can be efSdently deracemized to obtain the L-enantiomer using a multienzyme system consisting of D-amino acid oxidase, catalase, leucine dehydrogenase, and formate dehydrogenase. The a-keto acid 8 produced from the oxidation of the D-form is transformed into L-methionine 9 in the presence of ammonia, leucine dehydrogenase, and a stoichiometric amount of NADH. The NAD thus formed is recycled to NADH with ammonium formate and formate dehydrogenase [30] (Scheme 13.10). 

The coupling of these two enzymatic systems could find many more applications due to the avaUabihty of amino acid dehydrogenases of broader specificity [31]. A series of amino acid dehydrogenases with D-specificity for the preparation of D-amino acids has been applied to the reductive amination of a-keto acids [32]. However, the deracemization of rac-amino acids exploiting this type of enzyme requires an amino acid oxidase with L-specificily, which is a rare enzymatic activity. As an alternative the a-oxo acid, usually available through difficult synthetic procedures, can be used directly. 

The intermediate EiP, which is the major species of reduced enzyme with which O2 reacts in the amino acid oxidase reaction, is more reactive with O2 than Er in one case (49) (D-amino acid oxidase) but less reactive in the other (18) (n-amino acid oxidase). The reasons for such seemingly inconsistent behavior, as well as the virtual lack of reactivity of reduced flavins with O2 in systems such as succinic dehydrogenase, will only become clear when the molecular details of the oxidation mechanism of reduced flavin are elucidated. 

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