PDBsum entry 1t6d

Hydrolase

PDB id: 1t6d

Contents

Protein chains

303 a.a. *

Ligands

TRS

Metals

_CL ×2

Waters ×334

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural characterization of the stringent response related exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family.

Authors

O.Kristensen, M.Laurberg, A.Liljas, J.S.Kastrup, M.Gajhede.

Ref.

Biochemistry, 2004, 43, 8894-8900. [DOI no: 10.1021/bi049083c]

PubMed id

15248747

Abstract

Exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes play central roles in the bacterial stringent response induced by starvation. The high-resolution crystal structure of the putative Aquifex aeolicus PPX/GPPA phosphatase from the actin-like ATPase domain superfamily has been determined, providing the first insights to features of the common catalytic core of the PPX/GPPA family. The protein has a two-domain structure with an active site located in the interdomain cleft. Two crystal forms were investigated (type I and II) at resolutions of 1.53 and 2.15 A, respectively. This revealed a structural flexibility that has previously been described as a "butterfly-like" cleft opening around the active site in other actin-like superfamily proteins. A calcium ion is observed at the center of this region in type I crystals, substantiating that PPX/GPPA enzymes use metal ions for catalysis. Structural analysis suggests that nucleotides bind at a similar position to that seen in other members of the superfamily.

Secondary reference #1

Title

Role of inorganic polyphosphate in promoting ribosomal protein degradation by the lon protease in e. Coli.

Authors

A.Kuroda, K.Nomura, R.Ohtomo, J.Kato, T.Ikeda, N.Takiguchi, H.Ohtake, A.Kornberg.

Ref.

Science, 2001, 293, 705-708. [DOI no: 10.1126/science.1061315]

PubMed id

11474114

Figure 1.
Fig. 1. Growth lag of E. coli protease mutants after downshift and rates of protein turnover. (A) Escherichia coli MG1655 and its derivatives KY2966 (hslVU), KY2347 (lon, clpPX), and KY2350 (lon, clpPX, hslVU) (20) were grown to mid-log phase in an amino acid-rich medium and downshifted to a minimal medium (4). Growth was measured as the optical density at 600 nm (OD[600]) after the downshift. (B) Rates of intracellular protein degradation were measured (4); proteolysis was expressed as the percentage increase of the trichloroacetic acid-soluble radioactivity relative to the total incorporation (14C-leucine).

Figure 3.
Fig. 3. Degradation of ribosomal proteins by MBP-Lon or Lon in the presence of polyP in vitro and in vivo after the downshift. (A) A phospho-cellulose fraction (Fraction P9) obtained from the E. coli lysate (22) was incubated with 0.6 µg of MBP-Lon (11) in the presence of polyP (0.7 mM as P[i]; 1 µM as polymer). After incubation at 37°C for 60 min, exopolyphosphatase (yeast PPX, 3 × 10^4 U) was added to degrade the polyP; after 5 min, the mixture was subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and then visualized by silver staining (Di-ichi). (B) Fraction P9 was incubated with MBP-Lon (0.6 µg) in the presence of polyP (0.01, 0.1, or 1 µM) for 60 min and analyzed as described in (A). (C) Fraction P9 was incubated with Lon (1 µg) in the presence of polyP (1.4 µM). Samples were removed from the reaction mixture at the indicated times and then analyzed as in (A). Yeast PPX was not used. (D) PolyP with chain lengths of 65 and 15 residues (Sigma) was used for degradation of ribosomal proteins (23). The ribosomal proteins (2 µg) were incubated with MBP-Lon (0.1 µg) in the presence of polyP (0.64 mM as P[i]) for 60 min at 37°C and then analyzed as in (A). (E) S2-V5-epitope fusion (13) was expressed in the wild type and in the ppk mutant on 2× YT medium for 2 hours in the presence of 0.2% L-arabinose. Cells were collected by centrifugation (10 min, 3000g) and resuspended in the 2× YT medium without L-arabinose (No downshift) or in the MOPS minimal medium without L-arabinose (Downshift). At the indicated times, total proteins (100 µl of the culture) were subjected to SDS-PAGE and Western analysis with an antibody to V5 epitope (Invitrogen).

The above figures are reproduced from the cited reference with permission from the AAAs

Secondary reference #2

Title

Guanosine pentaphosphate phosphohydrolase of escherichia coli is a long-Chain exopolyphosphatase.

Authors

J.D.Keasling, L.Bertsch, A.Kornberg.

Ref.

Proc Natl Acad Sci U S A, 1993, 90, 7029-7033. [DOI no: 10.1073/pnas.90.15.7029]

PubMed id

8394006

Secondary reference #3

Title

Exopolyphosphate phosphatase and guanosine pentaphosphate phosphatase belong to the sugar kinase/actin/hsp 70 superfamily.

Authors

J.Reizer, A.Reizer, M.H.Saier, P.Bork, C.Sander.

Ref.

Trends Biochem Sci, 1993, 18, 247-248.

PubMed id

8212131