Racemase aspartate

Nimura N, Fujiwara T, Watanabe A, Sekine M, Furuchi T, et al. 2003. A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay. Anal Bio-chem 315 262-269. 

ASPARTYLCLUCOSAMINIDASE ASPARTATE AMINOTRANSFERASE ASPARTATE AMMONIA-LYASE ASPARTATE CARBAMOYLTRANSFERASE ASPARTATE a-DECARBOXYLASE ASPARTATE /3-DECARBOXYLASE ASPARTATE KINASE d-ASPARTATE OXIDASE ASPARTATE RACEMASE... 

The carboxyl group of an amino acid can also activate the a-hydrogen. This may be the basis for an aspartate racemase and other racemases that are not dependent upon PLP.156-158 See also Chapter 13, Section B,4. 

Aspartate a-decarboxylase 753, 755 Aspartate p-decarboxylase 746 Aspartate racemase 741 Aspartic acid (Asp, D) 52, 53s biosynthesis 517 pXa value of 293, 487 Aspartic proteases 621-625 Aspartyl aminopeptidase 621 p-Aspartyl phosphate 539, 540s Assays of enzyme activity 456 Assembly core of virus shell 365 Assembly pathway... 

Figure 12.11 Superposition of active sites from the different proteins. The active site of SmeHyuA model (gray) is compared with that of (a) aspartate racemase from Pyrococcus horikoshii (IF)LA, black) and that of (b) glutamate racemase from Aquifex pyrophilus (1B73A, black).

The procedure reported in Scheme 13.11 describes deracemization of an amino acid involving oxidation with an L-specific enzyme and transamination with a D-amino transferase using D-aspartate 10, which is generated from L-aspartate 11 by aspartate racemase, as the amino donor. The oxidative enzyme is defined as an L-amino acid deaminase, a flavoprotein from Proteus myxofadens [34]. The transamination reaction is shifted towards the product since the oxalacetate 12 formed decarboxylates spontaneously to give pyruvate and carbon dioxide. 

D-Amino acid transaminases have been less well characterized but proceed by a similar catalytic mechanism and show similar potential as effective biocatalysts. Further strain development has incorporated amino acid racemases, enabling complete utilization of racemic amino donors. Thus, L-aspartic acid can be used as the n-amino acid donor through use of aspartate racemase within the system. 

Okada et al. purified the enzyme to homogeneity from the cell extract of S. thermophilus, the specific activity of the crude extract of which was elevated 3400-fold1106. The gene encoding aspartate racemase was cloned from S. thermophilus, and overexpressed in E. colillll The amount of the enzyme produced reached... 

Aspartate racemase requires no cofactors and contains an essential cysteine residue in the same manner as glutamate racemase1801. When l- or D-aspartate was incubated with aspartate racemase in tritiated water, tritium was incorporated preferentially into the product enantiomer. This is consistent with the results of glutamate racemase as described above1911. 

Yamauchi et al.11121 concluded that aspartate racemase also uses two bases to remove and return the a-proton of the substrate. Aspartate racemase contains three cysteine residues Cys 84, Cys 190 and Cys 197, and only Cys 84 is essential for the enzyme activity. The alkylation of one cysteine residue/dimer with 2-nitro-5-thiocya-nobenzoic acid results in a complete loss of activity. Therefore, the enzyme shows a half-of-the-sites-reactivity11121. Yamauchi et al.11121 suggested that the enzyme has a composite active site formed at the interface of two identical subunits in the same manner as proposed for proline racemase1921. 

Kumagai and coworkers11131 developed an enzymatic procedure to produce d-alanine from fumarate by means of aspartase (E. C. 4.3.1.1), aspartate racemase, and D-amino acid aminotransferase (Fig. 17-12). Aspartase catalyzes conversion of fumarate into L-aspartate, which is racemized to form D-aspartate. D-Amino acid aminotransferase catalyzes transamination between D-aspartate and pyruvate to produce D-alanine and oxalacetate. This 2-oxo acid is easily decarboxylated spontaneously to form pyruvate in the presence of metals. Thus, the transamination proceeds exclusively toward the direction of D-alanine synthesis, and total conversion of fumarate into D-alanine was achieved. 

Figure 17-12. Enzymatic production of D-alanine by combination of aspartase, aspartate racemase, and D-amino acid aminotransferase reactions.

Sielaff, H. et al.. The mcyF gene of the microcystin biosynthetic gene cluster from Microcystis aeruginosa encodes an aspartate racemase, Biochem. J., 373, 909, 2003. 

M. Yohda, H. Okada and H. Kumagai, "Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus", Biochimica et Biophysica Acta 1089,234-240 (1991). 

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