Amino acid sequence of the L-lactate dehydrogenase of Bacillus caldotenax deduced from the nucleotide sequence of the cloned gene.
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 delta 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 SmaI 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.
Barstow
D A
DA
Murphy
J P
JP
Sharman
A F
AF
Clarke
A R
AR
Holbrook
J J
JJ
Atkinson
T
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D001411:Geobacillus stearothermophilus / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D007770:L-Lactate Dehydrogenase