Unique metal dependency of cytosolic alpha-mannosidase from Thermotoga maritima, a hyperthermophilic bacterium.
A putative cytosolic alpha-mannosidase gene from a hyperthermophilic marine bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The purified recombinant enzyme appeared to be a homodimer of a 110-kDa subunit. The enzyme showed metal-dependent ability to hydrolyze p-nitrophenyl-alpha-D-mannopyranoside. In the absence of a metal, the enzyme was inactive. Cobalt and cadmium supported high activity (60 U/mg at 70 degrees C), while the activity with zinc and chromium was poor. Cobalt (0.8 mol) bound to 1 mol monomer with a K(d) of 70 microM. The optimum pH and temperature were 6.0 and 80 degrees C, respectively. The activity was inhibited by swainsonine, but not by 1-deoxymannojirimycin, which is in agreement with the features of cytosolic alpha-mannosidase.
Nakajima
Masahiro
M
Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Imamura
Hiromi
H
Shoun
Hirofumi
H
Wakagi
Takayoshi
T
D000595:Amino Acid Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008358:Mannose / Q000737:chemistry; D008361:Mannosidases / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D043323:alpha-Mannosidase
D008670:Metals; D011994:Recombinant Proteins; D008361:Mannosidases; D043323:alpha-Mannosidase; D008358:Mannose