Characterization of a thermostable enzyme with phosphomannomutase/phosphoglucomutase activities from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.
The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.
Akutsu
Jun-ichi
J
National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki.
Zhang
Zilian
Z
Tsujimura
Masanari
M
Sasaki
Mayumi
M
Yohda
Masafumi
M
Kawarabayasi
Yutaka
Y
D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D002413:Cations, Divalent / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010733:Phosphoglucomutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D002413:Cations, Divalent; D008670:Metals; D011994:Recombinant Proteins; D017875:Phosphotransferases (Phosphomutases); D010733:Phosphoglucomutase; C052799:phosphomannomutase