Open AccessAmino acid sequence of the L‐lactate dehydrogenase of Bacillus caldotenax deduced from the nucleotide sequence of the cloned gene
Author(s) -
BARSTOW David A.,
MURPHY Jonathan P.,
SHARMAN Andy F.,
CLARKE Anthony R.,
HOLBROOK J. John,
ATKINSON Tony
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb11479.x
Subject(s) - nucleic acid sequence , biology , ribosomal binding site , microbiology and biotechnology , gene , peptide sequence , open reading frame , start codon , operon , structural gene , escherichia coli , biochemistry , homology (biology) , base pair , amino acid , stop codon , genetics , nucleotide , ribosome , rna
The Bacillus caldotenax L‐lactate dehydrogenase gene ( lct ) has been cloned into Escherichia coli , using the Bacillus stearothermophilus lct gene as a hybridisation probe, and its complete nucleotide sequence determined. The lct structural gene consists of an open reading frame of 951 base pairs commencing with an ATG start codon and followed by a TAA stop codon. Upstream of the gene are putative transcriptional promoter ‐35 and ‐10 regions; a ribosome binding site with a predicted Δ G of ‐66.9 kJ/mol is also present six base pairs upstream of the ATG start codon. The B. caldotenax lct gene is highly homologous to the B. stearothermophilus lct gene displaying a DNA sequence homology of 89.7%. Examination of the DNA sequence 3′ of the lct gene revealed the presence of two further open reading frames. This suggests that the lct gene may be the first gene of an operon. The deduced amino acid sequence of the L‐lactate dehydrogenase (LDH) from B. caldotenax predicted a protein of 317 amino acid residues; comparison with the B. stearothermophilus enzyme revealed only 30 amino acid differences between the two enzymes; thus the enzymes are 90.4% homologous. These amino acid differences must account for the different thermostabilities of the two enzymes. The B. caldotenax lct gene was efficiently expressed in E. coli and the original lct ‐containing plasmid construct isolated (pKD1) induced the synthesis of LDH at a level of 4.5% of the E. coli soluble cell protein whilst a Sma I subfragment of this clone, (pKD2) produced LDH at a level of 6.9% of the E. coli soluble cell protein. LDH isolated from E. coli cells had the same thermal stability properties as LDH isolated from B. caldotenax cells.