Examination of the structure, stability, and catalytic potential in the engineered phosphoryl carrier domain of pyruvate phosphate dikinase.


Pyruvate phosphate dikinase (PPDK) is a multidomain protein that catalyzes the interconversion of ATP, pyruvate, and phosphate with AMP, phosphoenolpyruvate (PEP), and pyrophosphate using its central domain to transport phosphoryl groups between two distant active sites. In this study, the mechanism by which the central domain moves between the two catalytic sites located on the N-terminal and C-terminal domains was probed by expressing this domain as an independent protein and measuring its structure, stability, and ability to catalyze the ATP/phosphate partial reaction in conjunction with the engineered N-terminal domain protein (residues 1-340 of the native PPDK). The encoding gene was engineered to express the central domain as residues 381-512 of the native PPDK. The central domain was purified and shown to be soluble, monomeric (13,438 Da), and stable (deltaG = 4.3 kcal/mol for unfolding in buffer at pH 7.0, 25 degrees C) and to possess native structure, as determined by multidimensional heteronuclear NMR analysis. The main chain structure of the central domain in solution aligns closely with that of the X-ray structure of native PPDK (the root-mean-square deviation is 2.2 A). Single turnover reactions of [14C]ATP and phosphate, carried out in the presence of equal concentrations of central domain and the N-terminal domain protein, did not produce the expected products, in contrast to efficient product formation observed for the N-terminal central domain construct (residues 1-553 of the native PPDK). These results are interpreted as evidence that the central domain, although solvent-compatible, must be tethered by the flexible linkers to the N-terminal domain for the productive domain-domain docking required for efficient catalysis. Study holds ProTherm entries: 19829 Extra Details: The PPDK central domain construct of residues 381-512; 0.1 mM EDTA and 1 mM DTT was added in the experiment phosphoryl groups; active sites; central domain construct; docking

Submission Details


Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Lin Y;Lusin JD;Ye D;Dunaway-Mariano D;Ames JB,Biochemistry (2006) Examination of the structure, stability, and catalytic potential in the engineered phosphoryl carrier domain of pyruvate phosphate dikinase. PMID:16460017
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1DIK 1995-12-06T00:00:00+0000 2.3 PYRUVATE PHOSPHATE DIKINASE
1GGO 2000-08-29T00:00:00+0000 2.6 T453A MUTANT OF PYRUVATE, PHOSPHATE DIKINASE
1JDE 2001-06-13T00:00:00+0000 2.8 K22A mutant of pyruvate, phosphate dikinase
1KBL 2001-11-06T00:00:00+0000 1.94 PYRUVATE PHOSPHATE DIKINASE
1KC7 2001-11-07T00:00:00+0000 2.2 Pyruvate Phosphate Dikinase with Bound Mg-phosphonopyruvate
2FM4 2006-01-06T00:00:00+0000 0 NMR structure of the phosphoryl carrier domain of pyruvate phosphate dikinase
2R82 2007-09-10T00:00:00+0000 3.6 Pyruvate phosphate dikinase (PPDK) triple mutant R219E/E271R/S262D adapts a second conformational state

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Pyruvate, phosphate dikinase P22983 PPDK_CLOSY