Tetrameric malate dehydrogenase from a thermophilic Bacillus: cloning, sequence and overexpression of the gene encoding the enzyme and isolation and characterization of the recombinant enzyme

摘要

pThe gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic iBacillus/i species (BI) has been cloned in an iEscherichia coli/i plasmid. The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa. Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic iBacillus/i species. Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs. MDHs and LDHs resemble each other in catalytic mechanism and several other respects. However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs. The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why iBacillus/i MDH, unlike most other MDHs, is tetrameric. A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression iE. coli/i plasmid vector. BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the iE. coli/i strain carrying the clone. MDH purified from BI and that purified from the iE. coli/i strain carrying the MDH gene clone appear to be identical proteins by several criteria. A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters. Whereas the MDH is a stable tetramer in the pH range 5–7, it appears to be converted into a stable dimer at pH 3.5. This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH./p