Dehydrogenase leucine

Similar to the work described by Spohn et al. [34], a trienzyme sensor was developed recently for the determination of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine). Leucine dehydrogenase, NADH oxidase, and peroxidase were coimmobilized covalently on tresylate-hydrophylic vinyl polymer beads and packed into a transparent PILL tube (20 cm X 1.0 id), which was used as flow cell. The sensor was free of interferences from protein and NH4+ and it was stable for 2 weeks. The sensor system was applied to the determination of branched-chain amino acids in plasma with recoveries ranging from 98 to 100% [36],... 

L-Leucine dehydrogenase in chiral L-amino acid synthesis 78... 

LEUCINE 2,3-AMINOMUTASE LEUCINE AMINOTRANSFERASE LEUCINE DEHYDROGENASE LEUCINE KINETICS LEUCYL-tRNA SYNTHETASE ZWITTERION yS-Leucine,... 

BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE LEUCINE DEHYDROGENASE LEUCINE KINETICS RROTEIN TURNOVER KINETICS SAAM... 

LEUCINE AMINOTRANSFERASE LEUCINE DEHYDROGENASE LEUCINE KINETICS... 

LACTATE DEHYDROGENASE LEUCINE DEHYDROGENASE LONG-CHAIN ALDEHYDE DEHYDROGENASE... 

Scheme 2.25 Kinetic resolution of racemic ferf-leucine 55 using leucine dehydrogenase.

An elegant four-enzyme cascade process was described by Nakajima et al. [28] for the deracemization of an a-amino acid (Scheme 6.13). It involved amine oxidase-catalyzed, (i )-selective oxidation of the amino acid to afford the ammonium salt of the a-keto acid and the unreacted (S)-enantiomer of the substrate. The keto acid then undergoes reductive amination, catalyzed by leucine dehydrogenase, to afford the (S)-amino acid. NADH cofactor regeneration is achieved with formate/FDH. The overall process affords the (S)-enantiomer in 95% yield and 99% e.e. from racemic starting material, formate and molecular oxygen, and the help of three enzymes in concert. A fourth enzyme, catalase, is added to decompose the hydrogen peroxide formed in the first step which otherwise would have a detrimental effect on the enzymes. 

L-tert-leucine trimethylpyruvate leucine dehydrogenase + formate dehydroenase... 

PEG-derivatised cofactor together with leucine, dehydrogenase is used in combination with the formate dehydrogenase required to regenerate the cofactor. 

Oka, M., Yang, Y.S., Nagata, S., Esaki, N., Tanaka, M., and Soda, K. 1989. Overproduction of thermostable leucine dehydrogenase of Bacillus stearothermophilus and its one-step purification from recombinant cells of Escherichia coli. Biotechnol. Appl. Biochem. 11 307-316. Sassenfeld, H.M. 1990. Engineering proteins for purification. Trends Biotechnol. 8 88-93. 

T. Ohshima, S. Nagata, and K. Soda, Purification and Characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus, Arch. Microbiol. 1985a, 141, 407 4-11. 

N. Esaki, and K. Soda, Construction of a new leucine dehydrogenase with preferred specificity for NADP+ by site-directed mutagenesis of the strictly NAD+-specific enzyme, Protein Eng. 1997, 10(6), 687-690. 

From the viewpoint of trying to find information on a particular enzyme, Figure 14.3 elucidates the possibilities with leucine dehydrogenase as an example. 

Accession number ] [Gene sequence] Alignment Modelling Secondary structure Figure 14.3 Information about an enzyme (e g., leucine dehydrogenase, LeuDH) in the databases. 

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... 

Hanson, R. L., Singh, I, Kissick, T. E, Patel, R. N., Szarka, L. I, and Mueller, R. H. 1990. Synthesis of L-P-hydroxyvaline from a-keto-P-hydroxyisovalerate using leucine dehydrogenase from Bacillus sp. Bioorg. Chem.,18,116-130. 

Figure 19.5. Cofactor regeneration systems for NAD(P)H-dependent enzyme reactions. The enzyme-coupled one involving GDH (a), that involving FDH (b), and the substrate-coupled one (c). AR1, aldehyde reductase from S. salmonicolor Leu DH, leucine dehydrogenase ADH, sec-alcohol dehydrogenase.

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