TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Members of Thermococcales harbor a number of genes encoding putative aminotransferase class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis. The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell surface glycoprotein gene TK0895 (P-csg). The purified protein did not display aminotransferase activity but exhibited racemase activity. An examination of most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (Delta TK1211) was constructed and grown on minimal medium supplemented with L- or D-Leu or L- or D-Met. The wildtype T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo. When we replaced L-Leu with D-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase but displayed cell yield similar to that observed in medium with L-Leu. In contrast, the Delta TK1211 strain displayed growth in medium with L-Leu but could not grow with D-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with L- or D-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensis. IMPORTANCE Phylogenetic analysis of aminotransferase class III proteins from all domains of life reveals numerous groups of protein sequences. One of these groups includes a large number of sequences from Thermococcales species and can be divided into four subgroups. Representatives of three of these subgroups have been characterized in detail. This study reveals that a representative from the remaining uncharacterized subgroup is an amino acid racemase with preference toward Leu and Met. Taken together with results of previous studies on enzymes from Pyrococcus horikoshii and Thermococcus kodakarensis, members of the four subgroups now can be presumed to function as a broad-substrate-specificity amino acid racemase (subgroup 1), alanine/serine racemase (subgroup 2), ornithine omega-aminotransferase (subgroup 3), or Leu/Met racemase (subgroup 4).

Automated summary

Members of Thermococcales contain genes encoding putative aminotransferase class III enzymes, with the TK1211 protein from Thermococcus kodakarensis exhibiting racemase activity towards Leu and Met. The TK1211 protein functions as a Leu racemase in vivo, as demonstrated by growth experiments with mutant strains on different Leu isomers. This study expands our understanding of the various activities of aminotransferase class III proteins across different subgroups.