Characterization of prolyl oligopeptidase from hyperthermophilic archaeon Thermococcus sp. NA1.
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 degrees C and 7, respectively. TNA1_POP activity appeared to be significantly activated by pre-incubation at 80 degrees C and 90 degrees 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 degrees C retaining 80% of its heat-activated activity even after 23 h, but it lost its enzymatic activity at 90 degrees C with a half-life of 3 h. The loss of the enzymatic activity at 90 degrees 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.
Lee
Hyun Sook
HS
Korea Ocean Research & Development Institute, Ansan P.O. Box 29, Seoul 425-600, Korea.
Kim
Yun Jae
YJ
Cho
Yona
Y
Kim
Sang-Jin
SJ
Lee
Jung-Hyun
JH
Kang
Sung Gyun
SG
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D019641:DNA, Archaeal / Q000235:genetics; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D000085822:Prolyl Oligopeptidases; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases