Open AccessPrimary Structure of Cyclohydrolase (Mch) from Methanobacterium thermoautotrophicum (Strain Marburg) and Functional Expression of the mch Gene in Escherichia coli
Author(s) -
Vaupel Martin,
Dietz Heiko,
Linder Dietmar,
Thauer Rudolf K.
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00294.x
Subject(s) - methanobacterium , gene , microbiology and biotechnology , biology , open reading frame , escherichia coli , primer extension , peptide sequence , gene expression , biochemistry , genetics , messenger rna , archaea
The gene mch encoding N 5 , N 10 ‐methenyltetrahydromethanopterin cyclohydrolase (Mch) in Methanobacterium thermoautotrophicum (strain Marburg) was cloned and sequenced. The gene, 963 bp, was found to be located at the 3′ end of a 3.5‐kbp Bam HI fragment. Upstream of the mch gene two open reading frames were recognized, one encoding for a 25‐kDa protein with sequence similarity to deoxyuridylate hydroxymethylase and the other encoding for a 34.6‐kDa protein with sequence similarity to cobalamin‐independent methionine synthase (MetE). The N‐terminal amino acid sequence deduced for the deoxyuridylate hydroxymethylase was identical to that previously published for thymidylate synthase ( Tys Y) from M. thermoautotrophicum. The 3′ end of the tys Y gene overlapped by 8 bp with the 5′ end of the mch gene. Despite this fact, the mch gene appeared to be transcribed monocistronically as evidenced by Northern blot analysis and primer‐extension experiments. The mch gene was overexpressed in Escherichia coli yielding an active enzyme of 37 kDa with a specific activity of 30 U/mg cell extract protein.