JBB : Vol. 103 (2007) , No. 3 p.221-228

Characterization of Prolyl Oligopeptidase from Hyperthermophilic Archaeon Thermococcus sp. NA1

Hyun Sook Lee¹⁾, Yun Jae Kim¹⁾, Yona Cho¹⁾, Sang-Jin Kim¹⁾, Jung-Hyun Lee¹⁾ and Sung Gyun Kang¹⁾

1) Korea Ocean Research & Development Institute

(Received 17-Oct-2006)
(Accepted 2-Dec-2006)

The prolyl oligopeptidase TNA1_POP was found to be encoded in the genome of the hyperthermophilic archaeon Thermococcus sp. NA1 and showed high similarities to its archaeal homologs (76–83%). The enzyme was found to be a single polypeptide composed of 616 amino acids with conserved signature domains. A recombinant TNA1_POP expressed in Escherichia coli was capable of hydrolyzing succinyl-Ala-Pro-p-nitroanilide (Suc-Ala-Pro-pNA) with temperature and pH optimums of 80°C and 7, respectively. TNA1_POP activity appeared to be significantly activated by pre-incubation at 80°C and 90°C with the optimum temperature unchanged. The heat-activated enzyme exhibited a k_cat approximately twofold higher than that of the unheated enzyme, however, both enzymes showed the same K_m. TNA1_POP was thermostable at 80°C retaining 80% of its heat-activated activity even after 23 h, but it lost its enzymatic activity at 90°C with a half-life of 3 h. The loss of the enzymatic activity at 90°C seemed to be caused by the autodegradation of the enzyme, not by thermal denaturation, as supported by circular dichroism spectropolarimetry. Autodegradation fragments ranging from 2 to 18 kDa were mapped by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

Key words: prolyl oligopeptidase, hyperthermophile, archaea, Thermococcus, autodegradation