Molecular Cloning, Expression, and Site-Directed Mutagenesis of Inorganic Pyrophosphatase from Thermus thermophilus HB8

Abstract

The genomic DNA encoding the inorganic pyrophosphatase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ATCC27634), was isolated by colony hybridization with a probe designed as a part of gene amplified by the PCR method, which was derived from the partial amino acid sequence of the enzyme. The DNA was cloned into a plasmid vector, pUC118, after digestion with BamHI. The inserted nucleotide fragment was about 1.8 kbp in length and the nucleotide sequence included a 525 bp open reading frame. The deduced amino acid sequence was completely identical with that of the enzyme determined by automated Edman analysis of peptide fragments isolated from digests obtained with Staphylococcus aureus V8 protease and Achromobacter protease I, and also from products obtained on chemical cleavage with cyanogen bromide and 70% formic acid. The subunit of this enzyme is composed of 174 amino acid residues with a calculated molecular weight of 19,084. Then, the gene was overexpressed in Escherichia coli BL21 (DE3) using a plasmid vector, pET15b, system. The recombinant enzyme was fully active, and exhibited higher thermostability than the E. coli enzyme. Amino acid residues located on the surface of the recombinant enzyme were determined by means of limited proteolysis, and the results revealed that the environment of Lys residues is almost the same as the crystal structure reported previously [Teplyakov, A. et al. (1994) Protein Sci. 3, 1098-1107]. Furthermore, the roles of two tryptophan residues were investigated by site-directed mutagenesis, which indicated that they may be responsible for the structural integrity and thermostability.

Evolutionary aspects of inorganic pyrophosphatase

“…When alternative PPase sequences with minor differences were found in the GenBank, only the sequences having the greatest similarity to the overall set were chosen, but the general conclusions presented below are valid for the alternative sequences as well. For PPases of S. cerevisiae [14], thermophilic bacterium PS‐3 [15], T. thermophilus [16] and Bacillus stearothermophilus [17], the primary structures have also been determined by protein sequencing and they are in good agreement with the gene‐deduced amino acid sequences.…”

Biotechnologically relevant enzymes from Thermus thermophilus

“…The pyrophosphatase from T. thermophilus was purified in a form free of non-specific RNAses and DNases (Satoh et al 1998). The enzyme eliminates organic pyrophosphate which is created during incorporation of nucleotide triphosphates.…”

“…Reduced activity is seen at 55°C (40%) and only residual activity remains at 45°C (20%). The gene from T. thermophilus over expressed in E. coli gave a fully active enzyme exhibiting higher thermostability than that of E. coli (Satoh et al 1998).…”

Cloning and expression of the inorganic pyrophosphatase gene from the amino acid producerBrevibacterium lactofermentumATCC 13869

“…Prokaryotic inorganic pyrophosphatase genes have been cloned from eubacteria ( Escherichia coli [4], Bacillus subtilis [5], Bartonella [6], Rhodospirillum [7]) and from Archaea ( Sulfolobus [8], Aquifex [9], Methanococcus [10], Thermus [11] and Thermoplasma [12]). When we attempted to characterise the proteins that interact with the essential cell division protein FtsZ from Brevibacterium lactofermentum , the PPase from this microorganism was purified.…”