Structure of the Full-length Enzyme I of the Phosphoenolpyruvate-dependent Sugar Phosphotransferase System

Abstract

Enzyme I (EI) is the phosphoenolpyruvate (PEP)-protein phosphotransferase at the entry point of the PEP-dependent sugar phosphotransferase system, which catalyzes carbohydrate uptake into bacterial cells. In the first step of this pathway EI phosphorylates the heat-stable phospho carrier protein at His-15 using PEP as a phosphoryl donor in a reaction that requires EI dimerization and autophosphorylation at His-190. The structure of the full-length protein from Staphylococcus carnosus at 2.5 Å reveals an extensive interaction surface between two molecules in adjacent asymmetric units. Structural comparison with related domains indicates that this surface represents the biochemically relevant contact area of dimeric EI. Each monomer has an extended configuration with the phosphohistidine and heat-stable phospho carrier protein-binding domains clearly separated from the C-terminal dimerization and PEP-binding region. The large distance of more than 35 Å between the active site His-190 and the PEP binding site suggests that large conformational changes must occur during the process of autophosphorylation, as has been proposed for the structurally related enzyme pyruvate phosphate dikinase. Our structure for the first time offers a framework to analyze a large amount of research in the context of the full-length model.Group translocation is the membrane transport mechanism by which a substrate is chemically modified to an impermeable derivative as it crosses the cell membrane. This energy-efficient transport strategy is used by bacteria for the uptake of rapidly metabolizable sugars, and it is achieved through a highly conserved three component phospho-relay system called the phosphoenolpyruvate:sugar phosphotransferase system (PTS) 4 (1-3). The PTS catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to a sugar while it is being transported across the membrane. It consists of two universal components, Enzyme I (molecular mass, 63 kDa) (4, 5), hereafter referred to as EI, and the heat-stable histidine phospho carrier protein (HPr) (molecular mass, 9 kDa), and in addition several membrane-associated components, which are sugar-specific and are collectively designated as Enzyme II (EII) complexes (3). The PTS cascade starts with the autophosphorylation of EI on a conserved histidine (His-190 in the Staphylococcus carnosus EI studied in this report) in a reaction that uses PEP as phosphoryl donor (4). Subsequently, the phosphoryl group is transferred to His-15 of the HPr protein, and ultimately to the imported hexose, in a series of transphosphorylation reactions mediated by the components of the sugar-specific EII complex. The PTS is not only responsible for sugar uptake; it also represents a major sensing and signaling system in the bacterial cell. The phosphorylation state of the components of the PTS pathway is directly coupled to the regulation of carbohydrate metabolism, chemotaxis toward carbon sources (6), carbon catabolite repression (7,8), and nitrogen metabolism (9). Because EI catalyze...

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein

“…While this article was being reviewed, the crystal structure of apo-EI from Staphylococcus carnosus was published (46). The His domain in this structure is largely disordered; however, the helical linker is defined and confirms our swiveling domain hypothesis.…”

Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

“…The first conformational state is optimal for the phosphotransfer reaction between EIN and HPr, and the second conformational state is relevant for the autophosphorylation reaction between EIN and EIC. [8][9][10] Isolated EIN adopts predominantly the first conformational state in solution, but the interaction with EIC requires a switch to the second conformational state. 5 Fig.…”

“…The linker region forms a long helix from residue Thr232 to residue Lys257, followed by a short b bridge (Ala261-Val269) that connects the linker helix to the (ab) 8 barrel fold of EIC. The EIC 231-575 construct contains the linker helix followed by EIC, whereas the EIC 260-575 construct lacks the whole linker helix.…”

“…The EIC 231-575 construct contains the linker helix followed by EIC, whereas the EIC 260-575 construct lacks the whole linker helix. Finally, the EIC 270-575 construct contains only the (ab) 8 barrel fold of EIC with both the linker helix and the b bridge removed. Figure 1(A) shows the three-dimensional structure of EI in a ribbon diagram, where EIN is colored in gray, EIC in blue, and the linker helix and the b bridge in red.…”

“…Crystal structures of EI have suggested the presence of multiple conformational states during the enzyme reactions, but it is not clear how the domain motions evolve with the substrate binding and phosphotransfer reactions. [8][9][10] Characterizing the interaction of isolated EIC with its ligands and also with EIN would provide useful information to understand the mechanism underlying the domain motions.…”