Sequencing, cloning, and high-level expression of the pfp gene, encoding a PP(i)-dependent phosphofructokinase from the extremely thermophilic eubacterium Dictyoglomus thermophilum

Abstract

The sequencing, cloning, and expression of the pfp gene from Dictyoglomus thermophilum, which consists of 1,041 bp and encodes a pyrophosphate-dependent phosphofructokinase, are described. A phylogenetic analysis indicates that the enzyme is closely related to the pyrophosphate-dependent enzyme from Thermoproteus tenax. The recombinant and native enzymes share a high degree of similarity for most properties examined.

FIG. 1

Multiple alignment of amino acid sequences of PFKs from D. thermophilum and nine other species, carried out by using Clustal W (version 1.6). B. stearother, B. stearothermophilus, pfp and pfk, genes encoding PP_i-PFK and ATP-PFK, respectively; A, F, and E, residues involved in binding ATP, F-6-P, and phosphoenolypyruvate (PEP), respectively, for the E. coli ATP-PFK. Asterisks, identical residues; dashes, gaps; dots, highly conserved residues.

FIG. 1

Multiple alignment of amino acid sequences of PFKs from D. thermophilum and nine other species, carried out by using Clustal W (version 1.6). B. stearother, B. stearothermophilus, pfp and pfk, genes encoding PP_i-PFK and ATP-PFK, respectively; A, F, and E, residues involved in binding ATP, F-6-P, and phosphoenolypyruvate (PEP), respectively, for the E. coli ATP-PFK. Asterisks, identical residues; dashes, gaps; dots, highly conserved residues.

FIG. 1

Multiple alignment of amino acid sequences of PFKs from D. thermophilum and nine other species, carried out by using Clustal W (version 1.6). B. stearother, B. stearothermophilus, pfp and pfk, genes encoding PP_i-PFK and ATP-PFK, respectively; A, F, and E, residues involved in binding ATP, F-6-P, and phosphoenolypyruvate (PEP), respectively, for the E. coli ATP-PFK. Asterisks, identical residues; dashes, gaps; dots, highly conserved residues.

FIG. 1

Multiple alignment of amino acid sequences of PFKs from D. thermophilum and nine other species, carried out by using Clustal W (version 1.6). B. stearother, B. stearothermophilus, pfp and pfk, genes encoding PP_i-PFK and ATP-PFK, respectively; A, F, and E, residues involved in binding ATP, F-6-P, and phosphoenolypyruvate (PEP), respectively, for the E. coli ATP-PFK. Asterisks, identical residues; dashes, gaps; dots, highly conserved residues.

FIG. 2

Phylogenetic relationships of PP_i- and ATP-PFKs. The tree is based on distance analysis (neighbor-joining method) of full amino acid sequences of PP_i- and ATP-PFKs in the EMBL and Swiss Prot databanks. Bootstrap values are based on 1,000 replicates and are given at each node. All of the PP_i-PFKs are bolded. Group I includes Thermus thermophilus, Thermotoga maritima (ATP-PFK), Aquifex aeolicus, Lactobacillus bulgaricus, B. stearothermophilus, and E. coli. Group II includes Treponema pallidum 0542, Borrelia burgdorferi 0020, Naegleria fowleri, Trichomonas vaginalis, Thermotoga maritima (PP_i-PFK), Treponema pallidum, B. burgdorferi, Trypanosoma brucei, Entamoeba histolytica, Propionibacterium freudenreichii, and Mycoplasma genitalium. Group III includes Thermoproteus tenax, D. thermophilum, Mycobacterium tuberculosis, A. methanolica, and S. coelicolor.

FIG. 3

SDS-PAGE gel (silver-stained) of fractions obtained during purification of the recombinant Dictyoglomus PP_i-PFK. Lane 1, molecular mass markers, as follows: phosphorylase b (molecular mass, 94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa); lane 2, cell extract following heat treatment; lanes 3 through 5, fractions obtained after purification through phenyl-Sepharose, Q-Sepharose, and red dye 120 ligand, respectively. Lanes 2, 3, 4, and 5 contained 3.0, 1.0, 0.75, and 0.75 μg of protein, respectively.