OVERPRODUCTION AND ONE-STEP PURIFICATION OF THE N-5,N-10-METHENYLTETRAHYDROMETHANOPTERIN CYCLOHYDROLASE (MCH) FROM THE HYPERTHERMOPHILIC METHANOPYRUS-KANDLERI

N-5,N-10-Methenyltetrahydromethanopterin cyclohydrolase (Mch) is an en zyme involved in methanogenesis from CO2 and H-2 which represents the energy metabolism of Methanopyrus kandleri, a methanogenic Archaeon gr owing at a temperature optimum of 98 degrees C. The gene mch from M. k andleri was cloned, sequenced, and expressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% in one step by chromatography on phenyl Sepharose in 80% ammonium sulfate. From 3. 5 g cells (250 mg protein); approximately 18 mg cyclohydrolase was obt ained. The purified enzyme showed essentially the same catalytic prope rties as the enzyme purified from M. kandleri cells. The primary struc ture and properties of the cyclohydrolase are compared with those of t he enzyme from Methanococcus jannaschii (growth temperature optimum 85 degrees C), from Methanobacterium thermoautotrophicum (65 degrees C), and from Methanosarcina barkeri (37 degrees C). Of the four enzymes, that from M. kandleri has the lowest isoelectric point (3.8) and the l owest hydrophobicity of amino acid composition. Besides, it has the hi ghest relative content of glutamate, leucine, and valine and the lowes t relative content of isoleucine, serine, and lysine. Some of these pr operties are unusual for enzymes from hyperthermophilic organisms. The y may reflect the observation that the cyclohydrolase from M. kandleri is not only adapted to hyperthermophilic conditions but also to the h igh intracellular concentrations of lyotrophic salts prevailing in thi s organism.