PRIMARY STRUCTURE OF CYCLOHYDROLASE (MCH) FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM (STRAIN MARBURG) AND FUNCTIONAL EXPRESSION OF THE MCH GENE IN ESCHERICHIA-COLI

The gene mch encoding N-5,N-10-methenyltetrahydromethanopterin cyclohy drolase (Mch) in Methanobacterium thermoautotrophicum (strain Marburg) was cloned and sequenced. The gene, 963 bp, was found to be located a t the 3' end of a 3.5-kbp BamHI fragment. Upstream of the mch gene two open reading frames were recognized, one encoding for a 25-kDa protei n with sequence similarity to deoxyuridylate hydroxymethylase and the other encoding for a 34.6-kDa protein with sequence similarity to coba lamin-independent methionine synthase (MetE). The N-terminal amino aci d sequence deduced for the deoxyuridylate hydroxymethylase was identic al to that previously published for thymidylate synthase (TysY) from M . thermoautotrophicum. The 3' end of the tysY gene overlapped by 8 bp with the 5' end of the mch gene. Despite this fact, the mch gene appea red to be transcribed monocistronically as evidenced by Northern blot analysis and primer-extension experiments. The mch gene was overexpres sed in Escherichia coli yielding an active enzyme of 37 kDa with a spe cific activity of 30 U/mg cell extract protein.