The first archaeal agmatinase from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization.
Agmatinase is one of the key enzymes in the biosynthesis of polyamines such as putrescine and sperimidine from arginine in microorganisms. The gene (PH0083) encoding the putative agmatinase of hyperthermophilic archaeon Pyrococcus horikoshii was identified based on the genome database. The gene was cloned and expressed, and the product was mainly obtained as inactive inclusion body in Escherichia coli. The inclusion body was dissolved in 6 M guanidine-HCl and successively refolded to active enzyme by the dilution of the denaturant. The enzyme exclusively catalyzed the hydrolysis of agmatine, but not arginine. This indicates that PH0083 codes agmatinase. The enzyme required divalent cations such as Co(2+), Ca(2+) and Mn(2+) for the activity. The highest activity was observed under fairly alkaline conditions, like pH 11. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of 110-145 kDa. The enzyme was extremely thermostable: the full activity was retained on heating at 80 degrees C for 10 min, and a half of the activity was retained by incubation at 90 degrees C for 10 min. From a typical Michaelis-Menten type kinetics, an apparent K(m) value for agmatine was determined to be 0.53 mM. Phylogenic analysis revealed that the agmatinase from P. horikoshii does not belong to any clusters of enzymes found in bacteria and eukarya. This is the first description of the presence of archaeal agmatinase and its characteristics.
Goda
Shuichiro
S
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Minamijosanjimacho, Tokushima 770-8506, Japan.
Sakuraba
Haruhiko
H
Kawarabayasi
Yutaka
Y
Ohshima
Toshihisa
T
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D003001:Cloning, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009952:Ornithine / Q000378:metabolism; D010802:Phylogeny; D011700:Putrescine / Q000378:metabolism; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D014512:Ureohydrolases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D001120:Arginine; D009952:Ornithine; D014512:Ureohydrolases; C498226:AGMAT protein, human; C019625:agmatinase; D011700:Putrescine