Amino formate dehydrogenase

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). 

In contrast, amino acid dehydrogenases comprise a well-known class of enzymes with industrial apphcations. An illustrative example is the Evonik (formerly Degussa) process for the synthesis of (S)-tert-leucine by reductive amination of trimethyl pyruvic acid (Scheme 6.12) [27]. The NADH cofactor is regenerated by coupling the reductive amination with FDH-catalyzed reduction of formate, which is added as the ammonium salt. 

The second E. coli example also involves contextual signals that alter coding patterns. A few proteins in all cells (such as formate dehydrogenase in bacteria and glutathione peroxidase in mammals) require the element selenium for their activity, generally in the form of the modified amino acid selenocysteine. Although modified amino acids are generally produced in posttranslational reactions (described in Section... 

Given an amino acid sequence, the secondary structure can be discerned with a program called psipred, which is available on the ExPASy site. Also, prediction of transmembrane location is possible nowadays. The procedure is illustrated in Figure 14.4 for formate dehydrogenase from Pseudomonas sp. 101 as an example. 

The element Se—not really a metal—is known to play a key role in enzymes such as the well-known glutathione peroxidase, formate dehydrogenase, glycine reductase, and the previously mentioned hydrogenases (Chapter 9). The unusual amino acid selenocysteine has a unique codon on the DNA (TGA/UGA), but selenation of serine also appears to be possible [16]. A brief review on Selenium can be found in the literature [17],... 

Formate dehydrogenase has been reported to have a pH optimum of 7.5-8.5 [25], The pH optimum for the reductive amination of (5) by an extract of T. intermedins was found to be about 8.7. Reductive amination reactions were carried out at pH 8.0. A summary of laboratory-scale batches is shown in Table 2. The time course for a representative batch showing conversion of ketoacid (5) to amino acid (4) is presented in Figure 5 using E. coli/C. boidinii heat-dried cells. 

Figure 5 Kinetics of enzymatic conversion of ketoacid acetal (5) to amino acid acetal (4) by phenylalanine dehydrogenase from recombinant It. coli and formate dehydrogenase from C. boidinii.

Phenylalanine dehydrogenase source Formate dehydrogenase source Keto acid (5) input (kg) Amino acid (4) output (kg) Reaction yield of (4) (M%) Enantiomeric excess amino acid (4) (%)... 

VO Popov, LA Shumilin, TB Ustinnikova, VS Lamzin, TA Egorov. NAD-dependent formate dehydrogenase from the methylotrophic bacterium Pseudomonas sp. 101. I. Amino acid sequence. Bioorg Khim 16 324-335, 1990. 

NIL,)02CH, dithiothreitol, formate dehydrogenase, TL 35 6533 (1994) amino acid dehydrogenase, NADH (enantioselec-tive, a-keto acid)... 

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). 

Reductions catalyzed by amino acid dehydrogenases formation of a-amino acids from ck -keto acids. 

Using an L-amino acid dehydrogenase in the presence of a formate dehydrogenase for cofactor regeneration, a prochiral keto acid is converted with high yield and en-antioselectivity. Furthermore, biocatalytic transaminations as well as Michael-addi-tions are important reactions for the large-scale synthesis of L-amino acids. 

The use of an LeuDH as an amino acid dehydrogenase showed a high L-enan-tiospecificity [24]. In this connection, an L-leucine dehydrogenase from Bacillus sphaericus has been applied very efficiently. The FDH from Candida boidinii is the preferred formate dehydrogenase for this process. The stability of this enzyme, which is available in technical quantities, has been remarkably improved by protein engineering and directed evolution [25], In particular the replacement of cys-... 

A very promising process route is the reductive amination of prochiral a-keto acids to a-amino acids with AADHs and the cofactor NADH and its regeneration by cooxidation of formate to CO2 by formate dehydrogenase (Fig. 15.3-1). 

Figure 16.7-8. Enzymatic deracemization of amino acids catalyzed by D-amino acid oxidase (d-AAO). Leucine dehydrogenase (LeuDH) transforms the oxidation product of the undesired amino acid enantiomer in situ into the racemic amino acid. Regeneration of NADH is performed by formate dehydrogenase (FHD).

Alanine racemase is a bacterial enzyme that catalyzes racemization of l- and d-alanine, and requires pyridoxal 5 -phosphate (PLP) as a cofactor. The enzyme plays an important role in the bacterial growth by providing D-alanine, a central molecule in the peptidoglycan assembly and cross-linking, and has been purified from various sources15 161. The enzyme has been used for the production of stereospecifically deuterated NADH and various D-amino acids by combination of L-alanine dehydrogenase (E. C. 1.4.1.1), D-amino acid aminotransferase (E. C. 2.6.1.21), and formate dehydrogenase (E.C. 1.2.1.2)I17, 18. ... 

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