Molecular cloning and enzymological characterization of pyridoxal 5′-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473

Abstract

We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5'-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on L-cysteic acid and L-cysteine sulfinic acid in addition to D- and L-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. D-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells.

A Novel Bifunctional Amino Acid Racemase With Multiple Substrate Specificity, MalY From Lactobacillus sakei LT-13: Genome-Based Identification and Enzymological Characterization

“…The standard assay conditions for analysis of Ls -MalY racemase activity were as follows: reaction mixture (1 mL) containing a 50 mM potassium phosphate (pH 7.5), 50 mM substrate, 50 μM PLP and Ls -MalY (200 μg), incubated at 30°C for 60 min. After stopping the reaction by boiling, the supernatant was subjected to high-performance liquid chromatography (HPLC) analysis, which was performed as described previously ( Gogami et al, 2011 ; Kato et al, 2015 ; Washio et al, 2016 ).…”

“…In general, amino acid racemase is classified into two groups: pyridoxal 5′-phosphate (PLP)-independent enzyme and PLP-dependent enzyme. The PLP-independent amino acid racemase includes glutamate racemase ( Choi et al, 1992 ; Yoshimura et al, 1993 ), aspartate racemase ( Fujii et al, 2015 ), and proline racemase ( Cardinale and Abeles, 1968 ) and contains two Cys residues as a catalytic residue ( Choi et al, 1992 ; Washio et al, 2016 ). In contrast, PLP-dependent amino acid racemases such as alanine racemase ( Oikawa et al, 2006 ) and arginine racemase ( Matsui et al, 2009 ) requires PLP as a cofactor.…”

Engineered Aminotransferase for the Production of d‐Phenylalanine Derivatives Using Biocatalytic Cascades

“…However, our cascades require stoichiometric amounts of the d ‐glutamate or d ‐aspartate donor substrate, which may represent a disadvantage for certain applications. This disadvantage can be eliminated by generating the donor substrate in situ with a glutamate or aspartate racemase and by replacing LAAD from Proteus mirabilis with that from Proteus myxofaciens , which has been shown to not deaminate l ‐glutamate or l ‐aspartate . Although we focused on the synthesis of d ‐phenylalanine derivatives in this study, other d ‐amino acids could also be synthesized using our cascades as all three DAAT variants reported here display increased activity relative to the wild type towards a series of d ‐amino acids that have aliphatic or polar side chains.…”

A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3

“…Free D -amino acids in cells are generally produced by amino acid racemases, which catalyze pyridoxal 5′-phosphate (PLP)-dependent or PLP-independent racemization of amino acids. PLP-independent Asp racemases (AspRs) and PLP-dependent AlaR and SerR have been found in archaeal cells and characterized ( Matsumoto et al, 1999 ; Long et al, 2001 ; Moore and Leigh, 2005 ; Ohnishi et al, 2008 ; Aihara et al, 2016 ; Washio et al, 2016 ). This includes detailed structural and functional characterization of a PLP-independent AspR from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 ( Liu et al, 2001 , 2002a , b ; Yoshida et al, 2006 ; Ohtaki et al, 2008 ; Kita et al, 2009 ).…”