The histidine containing phospho carrier protein (HPr) kinase͞phos-phatase is involved in carbon catabolite repression, mainly in Grampositive bacteria. It is a bifunctional enzyme that phosphorylates Ser-46-HPr in an ATP-dependent reaction and dephosphorylates P-Ser-46-HPr. X-ray analysis of the full-length crystalline enzyme from Staphylococcus xylosus at a resolution of 1.95 Å shows the enzyme to consist of two clearly separated domains that are assembled in a hexameric structure resembling a three-bladed propeller. The Nterminal domain has a ␣ fold similar to a segment from enzyme I of the sugar phosphotransferase system and to the uridyl-binding portion of MurF; it is structurally organized in three dimeric modules exposed to form the propeller blades. Two unexpected phosphate ions associated with highly conserved residues were found in the N-terminal dimeric interface. The C-terminal kinase domain is similar to that of the Lactobacillus casei enzyme and is assembled in six copies to form the compact central hub of the propeller. Beyond previously reported similarity with adenylate kinase, we suggest evolutionary relationship with phosphoenolpyruvate carboxykinase. In addition to a phosphate ion in the phosphate-binding loop of the kinase domain, we have identified a second phosphate-binding site that, by comparison with adenylate kinases, we believe accommodates a product͞substrate phosphate, normally covalently linked to Ser-46 of HPr. Thus, we propose that our structure represents a product͞substrate mimic of the kinase͞phosphatase reaction.signaling ͉ catabolite repression ͉ P-loop ͉ PEPCK ͉ AdK T he bacterial phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS) is a multicomponent transport system responsible for the uptake of carbohydrates. By using PEP as a primary phosphoryl group donor it catalyzes the phosphotransfer via the two general components enzyme I (EI) and histidine phospho carrier protein HPr to sugar specific enzyme II (EII) complex composed of hydrophilic domains EIIA and EIIB and the membrane-spanning domain EIIC (1). Besides its function as a group translocation system for carbohydrate uptake, PTS is a signaling device; it is linked to nitrogen metabolism (2), chemotaxis toward the sugar substrate (3), and additional regulatory functions (4).In enteric bacteria, the regulation of carbohydrate metabolism is mediated by EIIA glc of the PTS, which is the soluble part of the glucose-sensing membrane-associated EII complex (5, 6), whereas in Gram-positive bacteria, the regulation centers on the phosphocarrier protein HPr. HPr contains two phosphorylation sites. During the PEP-dependent phospho transfer, HPr is phosphorylated at His-15 by EI. In addition, HPr is subject to regulatory phosphorylation at Ser-46 by the ATP:Mg 2ϩ -dependent HPr kinase͞phos-phatase (HPrK), a general sensor for ATP, free phosphate (P i ), and glycolytic intermediates (7-10). The bifunctional enzyme has been identified in several Gram-positive microorganisms (7, 11-13) and some pathogenic b...
The interaction between the histidine-containing phosphocarrier protein HPr and xenon atoms in solution is studied in the present paper. Wild-type HPr as well as the exchange mutant I14A have been studied. Specific binding of xenon into an engineered cavity created via the exchange of amino acid residue I14 by alanine could be shown using 1H-15N heteronuclear single-quantum coherence (HSQC) spectroscopy. Xenon binding results in pronounced changes of the 1H and 15N chemical shifts of amide groups close to the cavity. In addition to this observation which allows the NMR-spectroscopic mapping of such cavities, we have shown that the entire molecule is slightly rearranged as a result of xenon binding. In contrast, wild-type HPr only exhibits minor chemical shift changes due to the nonspecific interactions with the xenon atoms in solution.
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