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 investigat ed in several hyperthermophiles, and the activity of dye-linked (L)-proline dehydrogenase (dye-(L)-proDH, (L)-proline:acceptor oxidoreductase) was fou nd in the crude extract of some Thermococcales strains. The enzyme was puri fied to homogeneity from a hyperthermophilic archaeon, Thermococcus profund us DSM 9503, which exhibited the highest specific activity in the crude ext ract. The molecular mass of the enzyme was about 160 kDa, and the enzyme co nsisted of heterotetrameric subunits (alpha (2) beta (2)) with two differen t molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequen ces of the alpha -subunit (50-kDa subunit) and the beta -subunit (40-kDa su bunit) were MRLT EHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respect ively. Dye-(L)-proDH was extraordinarily stable among the dye-linked dehydr ogenases under various conditions: the enzyme retained its full activity up on incubation at 70 degreesC for 10 min, and ca. 40% of the activity still remained after heating at 80 degreesC for 120 min. The enzyme did not lose the activity upon incubation over a nide range of pHs from 4.0 to 10.0 at 5 0 degreesC for 10 min. The enzyme exclusively catalyzed (L)-proline dehydro genation using 2,6-dichloroindophenol (CI2Ind) 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 grou p of the enzyme nas identified as flavin adenine dinucleotide by high-press ure liquid chromatography, In addition, the simple and specific determinati on of (L)-proline at concentrations from 0.10 to 2.5 mM using the stable dy e-(L)-proDH was achieved.