Sonia Fieulaine

Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: A relic of early life?

In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes (≈10% of their genome) by acting as catabolite corepressor. HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate. We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate. HprK/P, which binds phosphate at the same site as the β phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr. HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho–phosphorolysis mechanism. This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr. Growth of Bacillus subtilis on glucose increased intracellular pyrophosphate to concentrations (≈6 mM), which in in vitro tests allowed efficient pyrophosphate-dependent HPr phosphorylation. To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM. In B. subtilis, this might be achieved by YvoE. This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with hprK.

The HPr Kinase from Bacillus subtilis Is a Homo-oligomeric Enzyme Which Exhibits Strong Positive Cooperativity for Nucleotide and Fructose 1,6-Bisphosphate Binding

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2′(3′)-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.

Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism.

The oligomeric bifunctional HPr kinase/P‐Ser‐HPr phosphatase (HprK/P) regulates many metabolic functions in Gram‐positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C‐terminal region in contact with the ATP‐binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl‐phosphorylated HPr (P‐Ser‐HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P‐Ser‐HPr. The hprK V267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non‐PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non‐PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates.

X-ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain.

HPr kinase/phosphatase (HprK/P) is a key regulatory enzyme controlling carbon metabolism in Gram‐ positive bacteria. It catalyses the ATP‐dependent phosphorylation of Ser46 in HPr, a protein of the phosphotransferase system, and also its dephosphorylation. HprK/P is unrelated to eukaryotic protein kinases, but contains the Walker motif A characteristic of nucleotide‐binding proteins. We report here the X‐ray structure of an active fragment of Lactobacillus casei HprK/P at 2.8 Å resolution, solved by the multiwavelength anomalous dispersion method on a seleniated protein (PDB code 1jb1). The protein is a hexamer, with each subunit containing an ATP‐binding domain similar to nucleoside/nucleotide kinases, and a putative HPr‐binding domain unrelated to the substrate‐binding domains of other kinases. The Walker motif A forms a typical P‐loop which binds inorganic phosphate in the crystal. We modelled ATP binding by comparison with adenylate kinase, and designed a tentative model of the complex with HPr based on a docking simulation. The results confirm that HprK/P represents a new family of protein kinases, first identified in bacteria, but which may also have members in eukaryotes.

X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr

HPr kinase/phosphorylase (HprK/P) controls the phosphorylation state of the phosphocarrier protein HPr and regulates the utilization of carbon sources by Gram-positive bacteria. It catalyzes both the ATP-dependent phosphorylation of Ser-46 of HPr and its dephosphorylation by phosphorolysis. The latter reaction uses inorganic phosphate as substrate and produces pyrophosphate. We present here two crystal structures of a complex of the catalytic domain of Lactobacillus casei HprK/P with Bacillus subtilis HPr, both at 2.8-Å resolution. One of the structures was obtained in the presence of excess pyrophosphate, reversing the phosphorolysis reaction and contains serine-phosphorylated HPr. The complex has six HPr molecules bound to the hexameric kinase. Two adjacent enzyme subunits are in contact with each HPr molecule, one through its active site and the other through its C-terminal helix. In the complex with serine-phosphorylated HPr, a phosphate ion is in a position to perform a nucleophilic attack on the phosphoserine. Although the mechanism of the phosphorylation reaction resembles that of eukaryotic protein kinases, the dephosphorylation by inorganic phosphate is unique to the HprK/P family of kinases. This study provides the structure of a protein kinase in complex with its protein substrate, giving insights into the chemistry of the phospho-transfer reactions in both directions.

N-terminal protein modifications: Bringing back into play the ribosome

N-terminal protein modifications correspond to the first modifications which in principle any protein may undergo, before translation is completed by the ribosome. This class of essential modifications can have different nature or function and be catalyzed by a variety of dedicated enzymes. Here, we review the current state of the major N-terminal co-translational modifications, with a particular emphasis to their catalysts, which belong to metalloprotease and acyltransferase clans. The earliest of these modifications corresponds to the N-terminal methionine excision, an ubiquitous and essential process leading to the removal of the first methionine. N-alpha acetylation occurs also in all Kingdoms although its extent appears to be significantly increased in higher eukaryotes. Finally, N-myristoylation is a crucial pathway existing only in eukaryotes. Recent studies dealing on how some of these co-translational modifiers might work in close vicinity of the ribosome is starting to provide new information on when these modifications exactly take place on the elongating nascent chain and the interplay with other ribosome biogenesis factors taking in charge the nascent chains. Here a comprehensive overview of the recent advances in the field of N-terminal protein modifications is given.

The Structure of a Cyanobacterial Sucrose-Phosphatase Reveals the Sugar Tongs That Release Free Sucrose in the Cell

Sucrose-phosphatase (SPP) catalyzes the final step in the pathway of sucrose biosynthesis in both plants and cyanobacteria, and the SPPs from these two groups of organisms are closely related. We have crystallized the enzyme from the cyanobacterium Synechocystis sp PCC 6803 and determined its crystal structure alone and in complex with various ligands. The protein consists of a core domain containing the catalytic site and a smaller cap domain that contains a glucose binding site. Two flexible hinge loops link the two domains, forming a structure that resembles a pair of sugar tongs. The glucose binding site plays a major role in determining the enzymes remarkable substrate specificity and is also important for its inhibition by sucrose and glucose. It is proposed that the catalytic reaction is initiated by nucleophilic attack on the substrate by Asp9 and involves formation of a covalent phospho-Asp9-enzyme intermediate. From modeling based on the SPP structure, we predict that the noncatalytic SPP-like domain of the Synechocystis sucrose-phosphate synthase could bind sucrose-6F-phosphate and propose that this domain might be involved in metabolite channeling between the last two enzymes in the pathway of sucrose synthesis.

HPr Kinase/Phosphorylase, the Sensor Enzyme of Catabolite Repression in Gram-Positive Bacteria: Structural Aspects of the Enzyme and the Complex with Its Protein Substrate

HPr kinase/phosphorylase (HprK/P), the sensor enzyme for catabolite repression in gram-positive bacteria, phosphorylates HPr, a phosphocarrier protein of a sugar transport and phosphorylation system ([41][1]), at a serine residue ([10][2]). To carry out its catalytic function in sugar transport and

Structure of the full-length HPr kinase/phosphatase from Staphylococcus xylosus at 1.95 Å resolution: Mimicking the product/substrate of the phospho transfer reactions

The histidine containing phospho carrier protein (HPr) kinase/phosphatase is involved in carbon catabolite repression, mainly in Gram-positive 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 N-terminal domain has a βαβ 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 product/substrate phosphate, normally covalently linked to Ser-46 of HPr. Thus, we propose that our structure represents a product/substrate mimic of the kinase/phosphatase reaction.

Trapping conformational states along ligand-binding dynamics of peptide deformylase: the impact of induced fit on enzyme catalysis

For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally “zipped up” by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A “movie” that reconstructs this entire process can be further extrapolated to catalysis.