Molecular studies on lactobacillus casei lactate dehydrogenase gene

Abstract

A gene encoding allosteric L(+)-lactate dehydrogenase (LDH) (EC 1. 1.1.27) of $\underline{\mbox{Lactobacillus}}$ $\underline{\mbox{casei}}$ ATCC393 was cloned in $\underline{\mbox{Escherichia}}$ $\underline{\mbox{coli}}$ using oligonucleotide probes synthesized based on the previously determined amino acid sequence (R.Hensel, U.Mayr, and C.Y. Yang, Eur. J. Biochem., 134, 503-511, 1983). A complete nucleotide sequence of the gene was determined by the dideoxynucleotide method. The open reading frame comprising 981 base pairs (bp) started with a GTG and stopped with a TAA codon. The translated amino acid sequence agreed well with the previously determined amino acid sequence except for four exchanges at positions 38, 133, 281 and 285 as well as an inversion at 102 and 103. The inversion resulted in a replacement of Lys with Gln at position 102, which is highly conserved and responsible for the substrate discrimination. Sl nuclease mapping experiment revealed a couple of sequences for -35 and -10 region as CTGTCA and GATAAT, respectively, with a spacing of 18 nucleotides. The sequences of ribosome binding site were also found 10 bp upstream from the start codon as AGGA and AGAAAGGA identical to those of E. coli and $\underline{\mbox{B.}}$ $\underline{\mbox{subtilis}}$, respectively. Downstream from the TAA stop codon, there was a structure resembling $\rho$ -independent transcription terminator. The free energy of formation for the structure was -19.4 kcal. The allosteric LDH activity in E. coli was consistent throughout growth phase showing 12.7 U/mg protein when expressed solely by its own promoter and 123 U/mg protein when effected by the $\underline{\mbox{lac}}$ promoter of pUC19. In contrast, when transformed with the cloned $\underline{\mbox{ldh}}$ gene, $\underline{\mbox{B.}}$ $\underline{\mbox{subtilis}}$ produced the allosteric LDH in a growth-associated pattern as also shown in $\underline{\mbox{L.}}$ $\underline{\mbox{casei}}$. The maximum specific acti...