Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species

Abstract

Accumulation of D-leucine, D-allo-isoleucine, and D-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative ␥-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative ␥-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of L-isoleucine to D-allo-isoleucine and D-allo-isoleucine to L-isoleucine. In contrast, the enzyme showed no ␥-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K m and V max values of the enzyme for L-isoleucine were 5.00 mM and 153 mol·min ؊1 ·mg ؊1 , respectively, and those for D-allo-isoleucine were 13.2 mM and 286 mol·min ؊1 ·mg ؊1 , respectively. Hydroxylamine and other inhibitors of pyridoxal 5=-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5=-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.

Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by d-amino acids

“…Like the P. horikoshii BAR, the gene encoding the Ile 2-epimerase was originally annotated as a GABA-AT. The primary structure of Lactobacillus Ile 2-epimerase (450 residues) showed 40, 37, 36 and 44 % homology with those of P. horikoshii BAR (466 residues), PH0782 (474 residues), PH1423 (454 residues) and PH1501 (438 residues), respectively, all of which are classified as fold type I PLP-dependent enzymes (Grishin et al 1995;Mutaguchi et al 2013). These observations prompted us to speculate that the three other GABA-AT homologs may also function as racemases or epimerases.…”

“…Recently, a PLP-dependent Ile 2-epimerase was identified in Lactobacillus buchneri JCM 1115 as a novel AAR (Mutaguchi et al 2013). The Lactobacillus Ile 2-epimerase reacts with nonpolar amino acids such as Ile, Val and Leu as substrates, but not with Asp, Glu, Lys, Arg, His, Thr, Asn, Gln or Trp.…”

“…The crude extracts from P. horikoshii cultivated under various conditions were treated with 10 % trichloroacetic acid to precipitate the proteins and then neutralized with NaOH. Free amino acids in the resultant solution were derivatized with o-phthaldialdehyde (OPA) and N-acetyl-l-cysteine or with OPA and N-tert-butyloxycarbonyl-l-cysteine for diastereomerization (Aswad 1984;Hashimoto et al 1992;Mutaguchi et al 2013). The derivatives were then analyzed using the AccQ-Tag Ultra column on the UPLC system, as described by Mutaguchi et al (2013).…”

Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana

“…DAAR1 has similar enzymatic properties to other characterized amino acid racemases, both PLP-dependent and -independent. K m values between 2.5 and 110 mM have been reported for amino acid racemases (33,35,36,(38)(39)(40); the K m of DAAR1, at 17 mM, is within the expected range. It is interesting to note that Arabidopsis DAP-epimerase, a related enzyme involved in L-Lys biosynthesis, has a 100-fold lower K m value (23).…”

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

“…Recently, a second racemase belonging to AT-II was identified, namely isoleucine 2-epimerase [83]. This enzyme is quite peculiar not only because it is a racemase, but also because it operates on a standard α-amino acid substrate (together with D-FGAT and DGDA, it is one of the few AT-II enzymes whose preferred substrate is an α-amino acid rather than an ω-amine).…”