Formylmethanofuran:tetrahydromethanopterin formyltransferase (Ftr) from the hyperthermophilic Methanopyrus kandleri. Cloning, sequencing and functional expression of the ftr gene and one-step purification of the enzyme overproduced in Escherichia coli.
Methanopyrus kandleri is a methanogenic Archaeon that grows on H2 and CO2 at a temperature optimum of 98 degrees C. The gene ftr encoding the formylmethanofuran:tetrahydromethanopterin formyltransferase, an enzyme involved in CO2 reduction to methane, has been cloned, sequenced, and overexpressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% by simply heating the cell extract to 90 degrees C in 1.5 M K2HPO4 pH 8.0 for 30 min. From 1 g wet cells (70 mg protein) approximately 14 mg formyltransferase was obtained. The purified enzyme showed essentially the same catalytic properties as that purified from M. kandleri cells. The primary structure and properties of the formyltransferase are compared with those of the enzyme from Methanobacterium thermoautotrophicum (growth temperature optimum 65 degrees C) and Methanothermus fervidus (83 degrees C). Of the three enzymes that from M. kandleri had the lowest isoelectric point (4.2) and the lowest hydrophobicity of the amino acid composition. The enzyme from M. kandleri had the relatively highest content in alanine, glutamate and glutamine and the relatively lowest content in isoleucine, leucine and lysine. These properties, some of which are unusual for enzymes from other hyperthermophilic organisms, may reflect that the formyltransferase from M. kandleri is adapted to both hyperthermophilic and halophilic conditions.
Shima
S
S
Max-Planck-Institut für terrestrische Mikrobiologie, Philipps-Universität, Marburg, Germany.
Weiss
D S
DS
Thauer
R K
RK
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D019605:Euryarchaeota / Q000201:enzymology*; D019877:Hydroxymethyl and Formyl Transferases; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification; D014166:Transferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; C050840:formylmethanofuran-tetrahydromethanopterin formyltransferase