A D-amino acid dehydrogenase and an alanine racemase in a
hyperthermophile Pyrobaculum islandicum
Yoko Nagata, Mika Ito, Shigeru Toizaki, Takashi Sugizaki and Takaya Yamada
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University
1-8-14 Kanda, Surugadai, Chiyoda-Ku, Tokyo 101-8308 Japan
Fax: 03-3293-7572E-mail: nagata@chem.cst.nihon-u.ac.jp(Received 12 December 2002 Accepted 30 December 2002)
Abstract
Of archaea, methane-producing organisms do not possess D-enantiomers of amino acids.
contain D-serine and D-aspartic acid.

However, other archaea

In order to confirm the presence of D-amino acids in archaea, we cultivated thehyperthermophile cells at 95, and searched for enzyme activities of D-amino acid production.

Since a D-aminoacid-dehydrogenase activity and an alanine-racemase activity were detected, we partially purified these enzymes.

Theoptimum temperature of D-amino acid dehydrogenase was found to be 80, and the optimum pH was between 6.5 to9.0.

The enzyme activity was higher with D-proline as the substrate than with D-alanine.

was shown with both of the D- and L-alanine.

Alanine racemase activity

In addition, the racemase showed some activity with L-serine, although

the activity was less than 40% of that with L-alanine.

The optimum temperature of the racemase was 40 with L-alanine as substrate, but no temperature dependency of the enzyme was observed with D-alanine.

Key words: D-amino acid; D-amino acid dehydrogenase; racemase; hyperthermophile; archaea

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1.

Nagata, Y., Tanaka, K., Iida, T., Kera, Y., Yamada,R., Nakajima, Y., Fujiwara, T., Fukumori, Y.
Yamanaka, T., Koga, Y., Tsuji, S. and KawaguchiNagata, K. Occurrence of D-amino acids in a fewarchaea

anddehydrogenase

hyperthermophileactivities

Pyrobaculum

inislandicum,Biochimica et Biophysica Acta 1435, 160-166(1999).
2.

Kandler, O. and König, H. Arch. Microbiol. 118,141-152 (1978).

3.

Huber, R., Kristjansson, K., J. and Stetter, O, K.
Pyrobaculum

gen.

Nov.,

neutrophilic,rod-shaped

anewbyhigh-performanceliquidchromatography, Journal of Chromatography 575,

5/^ôWÇ4|dF

Nagata, Y., Yamamoto, K. and Shimojo, T.

genus

archaebacteria

offromcontinental solfataras growing optimally at 100À,Arch Microbiol 149, 95-101 (1987).

203