Exopolyphosphatases PPX1 and PPX2 from Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum accumulates up to 300 mM of inorganic polyphosphate (PolyP) in the cytosol or in granules. The gene products of cg0488 (ppx1) and cg1115 (ppx2) were shown to be active as exopolyphosphatases (PPX), as overexpression of either gene resulted in higher exopolyphosphatase activities in crude extracts and deletion of either gene with lower activities than those of the wild-type strain. PPX1 and PPX2 from C. glutamicum share only 25% identical amino acids and belong to different protein groups, which are distinct from enterobacterial, archaeal, and yeast exopolyphosphatases. In comparison to that in the wild type, more intracellular PolyP accumulated in the Deltappx1 and Deltappx2 deletion mutations but less when either ppx1 or ppx2 was overexpressed. When C. glutamicum was shifted from phosphate-rich to phosphate-limiting conditions, a growth advantage of the deletion mutants and a growth disadvantage of the overexpression strains compared to the wild type were observed. Growth experiments, exopolyphosphatase activities, and intracellular PolyP concentrations revealed PPX2 as being a major exopolyphosphatase from C. glutamicum. PPX2(His) was purified to homogeneity and shown to be active as a monomer. The enzyme required Mg2+ or Mn2+ cations but was inhibited by millimolar concentrations of Mg2+, Mn2+, and Ca2+. PPX2 from C. glutamicum was active with short-chain polyphosphates, even accepting pyrophosphate, and was inhibited by nucleoside triphosphates.

FIG. 1.

Exopolyphosphatase activity and PolyP accumulation in various strains of C. glutamicum. Open columns, exopolyphosphatase activity; filled columns, PolyP content expressed in millimolar P_i. Average values and standard deviations of at least three independent determinations are shown. Enzyme activity was measured spectrophotometrically using the EnzChek phosphate assay kit in 1 ml containing 50 mM PIPES, pH 6.8, 25 mM KCl, 2 mM MgCl₂, and 40 mM PolyP₂₀. *, P < 0.05; **, P < 0.005.

FIG. 2.

Purification of PPX2^His of C. glutamicum from E. coli BL21(DE3) (pET16b-ppx2). (Lane 1) SeaBlue Plus2 prestained standard (Invitrogen) containing proteins of the indicated molecular masses. Also shown is Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of protein from extracts before (lane 2) and 4 h after (lane 3) (both 150 μl OD₆₀₀⁻¹) induction with 0.5 mM IPTG and 12 μg PPX2^His purified by Ni-nitrilotriacetic acid chromatography (lane 4).

FIG. 3.

Influence of PolyP chain length on the activity of PPX2^His. For PolyP standards, see Materials and Methods. Enzyme activity was measured spectrophotometrically using the EnzChek phosphate assay kit in 1 ml containing 50 mM PIPES, pH 6.8, 25 mM KCl, 2 mM MgCl₂, up to 15 μg PPX2^His, and up to 40 mM PolyP.

FIG. 4.

Biomass formation (grams dry weight per liter) of C. glutamicum strains WT(pVWEx1), WT(pVWEx1-ppx1) and WT(pVWEx1-ppx2) (A and B), the WT, and the Δppx1 and Δppx2 strains (C and D), after transfer to minimal medium with different phosphate concentrations. Cells cultured overnight on LB medium were used to inoculate CgXII minimal medium with 4% (wt/vol) glucose and 0.13 mM (A and C) or 0 mM (B and D) phosphate. Cultivations shown in panels A and B were performed in the presence of 25 μg/ml kanamycin and 1 mM IPTG. Biomass formation was determined after 12 to 13 h of incubation. Averages and standard deviations of at least three independent cultivations are shown. *, P < 0.05; **, P < 0.005.

FIG. 5.

Phylogenetic tree of polyphosphatase (PPX, PPN), pyrophosphatase (PPA), and guanosine pentaphosphate phosphohydrolase (GPP) proteins and putative homologues. Numbers at the nodes represent bootstrap values. Full names of organisms are listed in Materials and Methods. Gene identifiers are shown after the names of organisms. Names of biochemically characterized enzymes are in parentheses. The positions of PPX1 and PPX2 of C. glutamicum are pointed out by arrows. M. smegmatis, Mycobacterium smegmatis; M. avium subsp. paratub., M. avium subspecies paratuberculosis.

FIG. 6.

Structure-based sequence alignment of the characterized exopolyphosphatases from E. coli, P. aeruginosa, and S. solfataricus with PPX1 and PPX2 from C. glutamicum and their homologues from M. tuberculosis, N. farcinica, S. coelicolor, and C. diphtheriae. The alignment was based on the crystal structure of E. coli PPX (37), and secondary structural motifs are highlighted above the alignment. Probable Mg²⁺ coordinating Asp143 and Glu150 are marked with asterisks. Putative catalytic Glu121 and Arg93 are highlighted with a circle and a square, respectively. Triangles indicate putative PolyP-binding canyons and diamonds indicate P-loop residues Gly145-Ser148. Gene identifiers are shown after the names of organisms. Sequence alignment was carried out using ClustalW, and the alignment was formatted using BoxShade.