Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.
Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95 °C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities.
National Institute of Advanced Industrial Science and Technology (AIST), Nakoji 3-11-46, Amagasaki, Hyogo, 661-0974, Japan.
Jun-ichi
J
Akutsu
State Key Laboratory of Marine Environmental Science, Institute of Marine Microbes and Ecospheres, Xiamen University, Xiamen, 361005, People's Republic of China.
Zilian
Z
Zhang
Laboratory on Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, Fukuoka, 812-8581, Japan.
Rihito
R
Morita
National Institute of Advanced Industrial Science and Technology (AIST), Nakoji 3-11-46, Amagasaki, Hyogo, 661-0974, Japan. kawarabayasi.yutaka@nifty.com.|Laboratory on Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, Fukuoka, 812-8581, Japan. kawarabayasi.yutaka@nifty.com.
Yutaka
Y
Kawarabayasi
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D008359:Mannose-6-Phosphate Isomerase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D043902:Pyrococcus horikoshii / Q000201:enzymology*
D019843:Archaeal Proteins; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; D008359:Mannose-6-Phosphate Isomerase
Genomic information; Hyperthermophilic archaea; Phosphohexose isomerase; Sugar-1-phosphate nucleotidylyltransferase; Thermostable enzyme