The rodA gene, which is responsible for the rod shape of Escherichia coli, was located 5 nucleotides downstream of another rod-shape-determining gene, pbpA, encoding penicillin-binding protein 2. The coding region for the RodA protein was 1,110 base pairs in length. Two plasmids, carrying a rodA-lacZ gene fusion with and without the pbpA promoter upstream of the gene fusion, were constructed. On the basis of the difference between the expression levels of the beta-galactosidase activity dependent on and independent of the pbpA promoter, we concluded that the pbpA and rodA genes constitute a single transcriptional unit called the rodA operon.
Thermus caldophilus GK24 was cloned in Escherichia coli using synthetic oligonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of the LDH was deduced from the nucleotide sequence. The deduced amino acid sequence agreed with the NH2-terminal and COOH-terminal sequences previously reported and the determined amino acid sequences of the peptides obtained from trypsin-digested T . caldophilus LDH. The LDH comprised 310 amino acid residues and its molecular mass was determined to be 32808. On alignment of the whole amino acid sequences, the T. caldophilus LDH showed about 40% identity with the Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle LDHs. The T. caldophilus LDH gene was expressed with the E. coli lac promoter in E. coli, which resulted in the production of the thermophilic LDH. The gene for the T . caldophilus LDH showed more than 40% identity with those for the human and mouse muscle LDHs on alignment of the whole nucleotide sequences.The G + C content of the coding regon for the T. caldophilus LDH was 74.1%, which was higher than that of the chromosomal DNA (67.2%). The G + C contents in the first, second and third positions of the codons used were 77.7%, 48.1% and 95.5% respectively. The high G + C content in the third base caused extremely non-random codon usage in the LDH gene. About half (48.7%) the codons in the LDH gene started with G, and hence there were relatively high contents of Val, Ala, Glu and Gly in the LDH. The contents of Pro, Arg, Ala and Gly, which have high G + C contents in their codons, were also high. Rare codons with U or A as the third base were sometimes used to avoid the TCGA sequence, the recognition site for the restriction endonuclease, Taql. Two TCGA sequences were found only in the sequence of CTCGAG (XhoI site) in the sequenced region of the T. caldophilus DNA. There were three segments with similar sequences in the two 5' non-coding regions, probably the promoter and ribosome-binding regions, of the genes for the T. caldophilus LDH and the Thermus thermophilus 3-isopropylmalate dehydrogenase.The structure and function of L-lactate dehydrogenases (LDHs) (L-lactate : NAD + oxidoreductase) from skeletal muscle and heart of vertebrates have been studied in great detail [l]. Vertebrate LDHs are non-allosteric enzymes, while some bacterial LDHs are known to be allosteric enzymes that are dependent on fructose 1,6-bisphosphate [2]. All these enzymes are tetramers in the active state, being composed of identical subunits with molecular masses of 30-35 kDa. The amino acid sequences of the allosteric LDHs from Luctobacillus casei [3] and Bacillus stearothermophilus [4] show high homology with those of non-allosteric vertebrate LDHs. The three-dimensional structure of the L. casei LDH is similar to those of vertebrate enzymes [5]. These findings indicate that
Fructose 1,6-bisphosphate (Fru-1,6-P,)-dependent L-lactate dehydrogenase (LDH) of Thermus culdophilus GK24 can be converted to a Fru-1,6-P,-independent form on modification with Arg-specific reagents. After trypsin digestion of the modified LDH, a peptide containing a modified Arg residue was purified and sequenced, and the modified Arg residue was identified as Arg-173. Subsequently, Arg-173 was replaced with Gln to remove the positive charge by site-directed mutagenesis.The mutant LDH was independent of Fru-1,6-P,, like non-allosteric vertebrate LDHs. Thus, Arg-173 was concluded to be located in the allosteric site and to be responsible for allosteric regulation of the LDH.
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