Histidine-containing Protein HPr Research Articles

Carbon catabolite repression in Thermoanaerobacterium saccharolyticum

BackgroundThe thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.ResultsWe report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.ConclusionCo-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

Visualization of Transient Ultra-Weak Protein Self-Association in Solution Using Paramagnetic Relaxation Enhancement

Ultra-weak macromolecular self-association is exceptionally difficult to both detect and visualize using conventional biophysical techniques owing to the very low population of the associated species, yet such weak intermolecular interactions coupled with nucleation events play an important role in driving spontaneous self-assembly to form higher-order architectures (such as crystals, viral capsids, and amyloid fibrils). In this article, we detect and characterize transient, ultra-weak self-association (KD >or= 15 mM) involving the histidine-containing protein HPr by means of paramagnetic relaxation enhancement (PRE), using EDTA-Mn2+ conjugated at three separate sites (E5C, E25C, and E32C, one at a time). Large intermolecular PRE effects, above the background observed with hydroxylamine-EDTA-Mn2+ as a control, are observed for two of the three paramagnetically labeled sites, E5C and E32C. The extent of self-association can be modulated (significantly reduced) by increasing the ionic strength or by the introduction of a negative charge (S46D mutation) within a positively charged surface patch, and abolished upon the addition of the N-terminal domain of enzyme I (EIN) to form a specific EIN-HPr complex. The PRE profiles observed for E5C and E32C can be fitted simultaneously and accounted for quantitatively using conjoined rigid body/torsion angle dynamics-simulated annealing with an ensemble of states to represent the distribution of one molecule of HPr relative to its partner.

Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.

Epitope mapping by mutagenesis distinguishes between the two tertiary structures of the histidine-containing protein HPr.

Thirty-four of the 85 residues of the histidine-containing protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system have been changed by site-directed mutagenesis. Many of the mutations have wild-type activity suggesting an unaltered tertiary structure but have altered binding to three monoclonal antibodies: Jel42, Jel44, and Jel323. This altered binding defines the residues that are involved in the epitopes of HPr. At present, two different three-dimensional structures have been determined for HPr, one from two-dimensional nuclear magnetic resonance spectra and the other from x-ray diffraction of HPr crystals. The epitope mapping for Jel42 does not distinguish between the tertiary structures. However, only the HPr structure derived from two-dimensional nuclear magnetic resonance spectra is consistent with a contiguous surface binding site that can be defined as the epitope for Jel44. Thus the x-ray structure may represent a partially unfolded HPr.

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli.

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.

Determination of the levels of HPr and enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium.

The levels of histidine-containing protein HPr and enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system of Escherichia coli strains 1100, NC3, W3110, and P650 and Salmonella typhimurium strains SB3507 and LJ144 have been determined by quantitative sugar phosphorylation assay and immunochemically. The levels have been determined for cells grown on minimal salts with glucose, fructose, mannitol, glycerol, and lactate and on nutrient broth. All determinations indicate a two- to three-fold change in the levels of enzyme I and HPr between growth on hexoses, which gave the higher levels, and the other growth substrates. The highest levels were not always found in glucose-grown cells. Antibodies were produced in rabbits using purified proteins from E. coli P650. The activity measurements and immunochemically determined enzyme I protein gave specific activities in the crude extracts of E. coli strains which were similar to that of the pure enzyme. The wild-type S. typhimurium enzyme I in crude extracts did not have the same immunochemical reactivity, although there was a considerable cross-reaction and the specific activity appeared to be half that of pure enzyme I. The HPr from both E. coli and S. typhimurium behaved identically and, although the immunoprecipitation was weak, it did indicate that HPr assays may not be as reliable as the enzyme I assays. The relative amounts of enzyme I and HPr found indicate that there are between 10- and 20-fold more HPr molecules in a cell than enzyme I subunits which form active dimers.