D-amino acid dehydrogenase

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D-amino-acid dehydrogenase (EC 1.4.99.1) is a bacterial enzyme that catalyses the oxidation of D-amino acids into their corresponding oxoacids. It contains both flavin and nonheme iron as cofactors. [1] The enzyme has a very broad specificity and can act on most D-amino acids. [2]

Contents

D-amino acid + H2O + acceptor <=> a 2-oxo acid + NH3 + reduced acceptor

This reaction is distinct from the oxidation reaction catalysed by D-amino acid oxidase that uses oxygen as a second substrate, as the dehydrogenase can use many different compounds as electron acceptors, with the physiological substrate being coenzyme Q. [1] [3]

D-amino acid dehydrogenase is an enzyme that catalyzes NADPH from NADP+ and D- glucose to produce D- amino acids and glucose dehydrogenase. Some but not limited to these amino acids are D-leucine, D-isoleucine, and D-Valine, which are essential amino acids that humans cannot synthesize due to the fact that they are not included in their diet. Moreover, D- amino acids catalyzes the formation of 2-oxo acids to produce D- amino acids in the presence of DCIP which is an electron acceptor. [4] D-amino acids are used as components of pharmaceutical products, such as antibiotics, anticoagulants, and pesticides, because they have been shown to be not only more potent than their L enantiomers, but also more resistant to enzyme degradation. [5] D-amino acid dehydrogenase enzymes have been synthesized via mutagenesis with an ability to produce straight, branched, cyclic aliphatic and aromatic D-amino acids. [5] Solubilized D-amino acid dehydrogenase tends to increase its affinity for D-alanine, D-asparagine, and D--amino-n-butyrate. [6]

In E. coli K12 D-amino acid dehydrogenase is most active with D-alanine as its substrate, as this amino acid is the sole source of carbon, nitrogen, and energy. The enzyme works optimally at pH 8.9 and has a Michaelis constant for D-alanine equal to 30 mM. [7] DAD discovered in gram-negative E. coli B membrane can convert L-amino acids into D-amino acids as well. [8]

Additionally, D- amino acid dehydrogenase is used in Dye-Linked dehydrogenase (Dye-DHs) which uses artificial dyes such as 2,6-Dichloroindophenol (DCIP) as their electron acceptor rather than using their natural electron acceptors. This can accelerate the reaction between the enzyme and the substrate when the electrons are being transferred. [9]

Use in synthesis reactions

D-Amino Acid Dehydrogenase has shown itself to be effective in the synthesis of branched-chain amino acids such as D-Leucine, D-Isoleucine, and D-Valine. In the given study, researchers were successfully able to use D-amino acid dehydrogenase to create high amounts of these products from the starting material of 2-oxo acids, in the presence of ammonia. The conditions for this were variable, though the best results appeared at around 65 °C.

Amino Acids obtained through these reactions resulted in a high enantioselectivity of >99% and high yields of >99%.

Given the nature of this enzyme, it may be possible to use it in order to create non-branched D-amino acids as well as modified D-amino acids. [10]

Obtaining D-Amino Acid Dehydrogenase

In one study, in order to test the viability of using D-amino dehydrogenase in synthesis reactions, researchers used mutant bacteria to obtain and create different strains of the enzyme. These researchers found that it only required five mutations in order to modify the selective D-Amino Dehydrogenase into working with other D-amino acids. They also found that it retained its highly selective nature, capable of receiving mostly D-enantiomers after mutation, with yields in excess of 95%. [5]

A heat-stable variant of D-amino acid dehydrogenase was found in the bacterium Rhodothermus marinus JCM9785. This variant is involved in the catabolism of trans-4-hydroxy-L-proline. [11]

From the given studies, in order to obtain D-amino acid dehydrogenase one must first introduce and express it within a given bacterial species, some of which have been previously referenced. It must then be purified under favorable conditions. These are based upon the particular species of D-amino acid dehydrogenase used in a given research experiment. Under incorrect conditions, the protein may denature. For example, it was found that specifically D-alanine dehydrogenases from E. coli and P. aeruginosa would lose most of their activity when subjected to conditions of 37 - 42 °C. After this, it is possible to separate and purify through existing methods. [12]

Artificial D-Amino Acid Dehydrogenase

Due to the drawbacks of current methods, researchers have begun work on creating an artificial enzyme capable of producing the same D-amino acids as enzymes from naturally occurring sources. By adding five amino acids to a given sample isolated from U. thermosphaericus, they succeeded. By modifying the amino acid sequence, researchers were able to change the specificity of the molecule towards certain reactants and products, showing that it may be possible to use artificial D-amino acid dehydrogenase to screen for certain D-amino acid products. [13]

See also

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References

  1. 1 2 Olsiewski PJ, Kaczorowski GJ, Walsh C (25 May 1980). "Purification and properties of D-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur flavoenzyme from Escherichia coli B". J. Biol. Chem. 255 (10): 4487–94. doi: 10.1016/S0021-9258(19)85517-5 . PMID   6102989.
  2. ↑ Tsukada K (10 October 1966). "D-amino acid dehydrogenases of Pseudomonas fluorescens". J. Biol. Chem. 241 (19): 4522–8. doi: 10.1016/S0021-9258(18)99750-4 . hdl: 2433/211070 . PMID   5925166.
  3. ↑ Jones H, Venables WA (1983). "Effects of solubilisation on some properties of the membrane-bound respiratory enzyme D-amino acid dehydrogenase of Escherichia coli". FEBS Letters. 151 (2): 189–92. doi: 10.1016/0014-5793(83)80066-0 . PMID   6131836. S2CID   26120769.
  4. ↑ Akita, Hironaga; Suzuki, Hirokazu; Doi, Katsumi; Ohshima, Toshihisa (1 February 2014). "Efficient synthesis of d-branched-chain amino acids and their labeled compounds with stable isotopes using d-amino acid dehydrogenase". Applied Microbiology and Biotechnology. 98 (3): 1135–1143. doi:10.1007/s00253-013-4902-1. ISSN   1432-0614. PMID   23661083. S2CID   2640504.
  5. 1 2 3 Vedha-Peters, Kavitha; Gunawardana, Manjula; Rozzell, J. David; Novick, Scott J. (August 2006). "Creation of a Broad-Range and Highly Stereoselectived-Amino Acid Dehydrogenase for the One-Step Synthesis ofd-Amino Acids". Journal of the American Chemical Society. 128 (33): 10923–10929. doi:10.1021/ja0603960. ISSN   0002-7863. PMC   2533268 . PMID   16910688.
  6. ↑ Jones, H (January 1983). "Effects of solubilisation on some properties of the membranebound respiratory enzyme D-amino acid Escherichia coli". FEBS Letters. 151 (2): 189–192. doi: 10.1016/0014-5793(83)80066-0 . PMID   6131836. S2CID   26120769.
  7. ↑ Franklin, F.C.H. (10 January 1976). "Biochemical, genetic, and regulatory studies of alanine catabolism in Escherichia coli K12". Molecular and General Genetics. 149 (2): 229–237. doi:10.1007/BF00332894. PMID   13292. S2CID   22823588.
  8. ↑ Xu, Jinjin; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie (4 July 2017). "Expression, purification, and characterization of a membrane bound d-amino acid dehydrogenase from Proteus mirabilis JN458". Biotechnology Letters. 39 (10): 1559–1566. doi:10.1007/s10529-017-2388-0. ISSN   0141-5492. PMID   28676939. S2CID   11335026.
  9. ↑ Satomura, Takenori; Sakuraba, Haruhiko; Suye, Shin-ichiro; Ohshima, Toshihisa (1 November 2015). "Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology". Applied Microbiology and Biotechnology. 99 (22): 9337–9347. doi:10.1007/s00253-015-6944-z. ISSN   1432-0614. PMID   26362681. S2CID   12495865.
  10. ↑ Akita, Hironaga; Suzuki, Hirokazu; Doi, Katsumi; Ohshima, Toshihisa (2013-05-10). "Efficient synthesis of d-branched-chain amino acids and their labeled compounds with stable isotopes using d-amino acid dehydrogenase". Applied Microbiology and Biotechnology. 98 (3): 1135–1143. doi:10.1007/s00253-013-4902-1. ISSN   0175-7598. PMID   23661083. S2CID   2640504.
  11. ↑ Satomura, Takenori; Ishikura, Masaru; Koyanagi, Takashi; Sakuraba, Haruhiko; Ohshima, Toshihisa; Suye, Shin-ichiro (2014-12-05). "Dye-linked d-amino acid dehydrogenase from the thermophilic bacterium Rhodothermus marinus JCM9785: characteristics and role in trans-4-hydroxy-l-proline catabolism". Applied Microbiology and Biotechnology. 99 (10): 4265–4275. doi:10.1007/s00253-014-6263-9. ISSN   0175-7598. PMID   25472442. S2CID   11805375.
  12. ↑ Xu, Jinjin; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie (2017-07-04). "Expression, purification, and characterization of a membrane-bound d-amino acid dehydrogenase from Proteus mirabilis JN458". Biotechnology Letters. 39 (10): 1559–1566. doi:10.1007/s10529-017-2388-0. ISSN   0141-5492. PMID   28676939. S2CID   11335026.
  13. ↑ Akita, Hironaga; Hayashi, Junji; Sakuraba, Haruhiko; Ohshima, Toshihisa (2018-08-03). "Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application". Frontiers in Microbiology. 9: 1760. doi: 10.3389/fmicb.2018.01760 . ISSN   1664-302X. PMC   6085447 . PMID   30123202.
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