Purification, characterization, and application of a novel dye-linked L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus.
The distribution of dye-linked L-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked L-proline dehydrogenase (dye-L-proDH, L-proline:acceptor oxidoreductase) was found in the crude extract of some Thermococcales strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon, Thermococcus profundus DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (alpha(2) beta(2)) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the alpha-subunit (50-kDa subunit) and the beta-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-L-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70 degrees C for 10 min, and ca. 40% of the activity still remained after heating at 80 degrees C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50 degrees C for 10 min. The enzyme exclusively catalyzed L-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for L-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Delta(1)-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of L-proline at concentrations from 0.10 to 2.5 mM using the stable dye-L-proDH was achieved.
Sakuraba
H
H
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjimacho, Tokushima 770-8506, Japan.
Takamatsu
Y
Y
Satomura
T
T
Kawakami
R
R
Ohshima
T
T
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000595:Amino Acid Sequence; D004396:Coloring Agents / Q000737:chemistry; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism; D011394:Proline Oxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology* / Q000254:growth & development
D004396:Coloring Agents; D011392:Proline; D015086:2,6-Dichloroindophenol; D011394:Proline Oxidase