D-Amino Acids and Amino Acid Racemases in Hyperthermophilic Archaea
Masafumi YOHDA*a, Yuko AIKAWAa, Isao ENDOa, Zhiqun LONGb, Hiroshi HOMMAb, Kazuhiro IMAIb, Toshii IIDAc, and Tadashi MARUYAMAc
a Biochemical Systems Laboratory, The Institute of Physical and Chemical Research, Wako, Saitama 351-01, Japan
b Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
c Marine Biotechnology Institute, Kamaishi, Iwate 026, Japan
D-Amino acids are important components of eubacteria, as they constitute parts of the peptide chains in murein of cell walls (1). D-Alanine is formed from L-alanine by alanine racemase that is quite widespread in eubacteria. Other D-amino acids, such as D-aspartate or D-glutamate, are synthesized either by transamination from D-alanine to the appropriate α-keto acid or by racemization of the enantiomeric L-amino acid.
There are few reports describing endogenous D-amino acids in eukaryotes or archaea. Although several groups have identified D-serine and D-aspartate in mammals, the biosynthetic pathway of these D-amino acids is unknown (2,3). Murein is the only cell wall polymer in eubacteria. On the contrary, archaea possess a broader range of cell envelope structural formats and their cell walls do not contain murein (4). Cell envelopes of some archaea, including methanogens, consist of pseudomurein whose structure is homologous to murein. However, pseudomurein does not contain D-amino acids in its peptide unit. Thus, it had been believed that there were neither D- amino acids nor amino acid racemases in archaea. However, recently, we have cloned a gene coding aspartate racemase form the sulfur-dependent hyperthermophilic archaeum Desulfurococcus strain SY (5). We have also detected small aspartate racemazing activity in the crude extract of the strain.
To investigate whether amino acid racemases or D-amino acids are widely distributed among archaea, we have searched them in several other archaea. PCR amplification was performed using the primers designed on consensus sequences among aspartate racemase genes so far sequenced, and we could obtain DNA fragments highly homologous to Desulfurococcus strain SY aspartate racemase gene from several Thermococcus strains that were isolated in Kodakara-jima island. The cloning of the full-length aspartate racemase genes from these strains is under progress. We have also detected the existence of D-aspartate in Desulfurococcus strain SY and also in Thermococcus strain KS8. D-Forms of other amino acids were also detected in these strains.
Although the functions of amino acid racemases or D-amino acids in archaea are
