@article{BiswasKayasthaSeckler2003,
  author    = {Biswas, Shyamasri and Kayastha, Arvind M. and Seckler, Robert},
  title     = {Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14},
  issn      = {0176-1617},
  year      = {2003},
  abstract  = {Summary Using five different steps, ;-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeniety with approximately 90-fold purificationwith a specific activity of 281 units mg;1 protein. A single bandwas observed in native PAGE. Activity staining of the native gel with 5-bromo4-chloro 3-indoxyl ;-D-galactopyranoside (X-Gal) at pH 4.0 also produceda single band. Analytical gel filtration in Superdex G-75 revealed the molecularmass of the native protein to be approximately 75 kD. 10 percnt; SDS-PAGE under reducingconditions showed two subunits of molecular masses, 45 and 30 kD, respectively.Hence, ;-galactosidase from kidney beans is a heterodimer. A typical proteinprofile with ;max at 280 nm was observed and A280/A260ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86 percnt; sequencehomology with an Arabidopsis thaliana and 85 percnt; with Lycopersiconesculentum putative ;-galactosidase sequences. The Electrospray MassSpectrometric analysis of this band also revealed a peptide fragment that had90 percnt; sequence homology with an Arabidopsis thaliana putative ;- galactosidasesequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometricanalysis both by MALDI- TOF and ES MS revealed certain sequences that matchedwith phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0and it hydrolysed o- and p-nitrophenyl ;-D galactopyranosidewith a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively.The energy of activation calculated from the Arrhenius equation was 14.8 kcal/molenzyme site. The enzyme was found to be comparatively thermostable showing maximumactivity at 67 °C. Thermal denaturation of the enzyme at 65 °C obeyssingle exponential decay with first order-rate constant 0.105 min;1.Galactose, a hydrolytic product of this enzyme was a competitive inhibitor witha Ki of 2.7 mmol/L.},
  language  = {en}
}
@article{BaxaWeintraubSeckler2020,
  author    = {Baxa, Ulrich and Weintraub, Andrej and Seckler, Robert},
  title     = {Self-competitive inhibition of the bacteriophage P22 Tailspike endorhamnosidase by O-antigen oligosaccharides},
  series = {Biochemistry},
  volume    = {59},
  journal   = {Biochemistry},
  number    = {51},
  publisher = {American Chemical Society},
  address   = {Washington},
  issn      = {0006-2960},
  doi       = {10.1021/acs.biochem.0c00872},
  pages     = {4845 -- 4855},
  year      = {2020},
  abstract  = {The P22 tailspike endorhamnosidase confers the high specificity of bacteriophage P22 for some serogroups of Salmonella differing only slightly in their O-antigen polysaccharide. We used several biophysical methods to study the binding and hydrolysis of O-antigen fragments of different lengths by P22 tailspike protein. O-Antigen saccharides of defined length labeled with fluorophors could be purified with higher resolution than previously possible. Small amounts of naturally occurring variations of 0antigen fragments missing the nonreducing terminal galactose could be used to determine the contribution of this part to the free energy of binding to be similar to 7 kJ/mol. We were able to show via several independent lines of evidence that an unproductive binding mode is highly favored in binding over all other possible binding modes leading to hydrolysis. This is true even under circumstances under which the O-antigen fragment is long enough to be cleaved efficiently by the enzyme. The high-affinity unproductive binding mode results in a strong self-competitive inhibition in addition to product inhibition observed for this system. Self-competitive inhibition is observed for all substrates that have a free reducing end rhamnose. Naturally occurring O-antigen, while still attached to the bacterial outer membrane, does not have a free reducing end and therefore does not perform self-competitive inhibition.},
  language  = {en}
}
@article{BaxaSteinbacherWeintraubetal.1999,
  author    = {Baxa, Ulrich and Steinbacher, Stefan and Weintraub, Andrej and Huber, Robert and Seckler, Robert},
  title     = {Mutations improving the folding of phage P22 tailspike protein affect its receptor binfing activity},
  year      = {1999},
  language  = {en}
}
@article{BaxaCooperWeintraubetal.2001,
  author    = {Baxa, Ulrich and Cooper, Alan and Weintraub, N. and Pfeil, Wolfgang and Seckler, Robert},
  title     = {Enthalpic barriers to the hydrophobic binding of oligosaccharides to phage P22 tailspike protein},
  year      = {2001},
  language  = {en}
}
@article{BarbirzMuellerUetrechtetal.2008,
  author    = {Barbirz, Stefanie and M{\"u}ller, J{\"u}rgen J. and Uetrecht, Charlotte and Clark, Alvin J. and Heinemann, Udo and Seckler, Robert},
  title     = {Crystal structure of Escherichia coli phage HK620 tailspike : podoviral tailspike endoglycosidase modules are evolutionarily related},
  issn      = {0950-382X},
  year      = {2008},
  abstract  = {Bacteriophage HK620 infects Escherichia coli H and is closely related to Shigella phage Sf6 and Salmonella phage P22. All three Podoviridae recognize and cleave their respective host cell receptor polysaccharide by homotrimeric tailspike proteins. The three proteins exhibit high sequence identity in the 110 residues of their N-terminal particle- binding domains, but no apparent sequence similarity in their major, receptor-binding parts. We have biochemically characterized the receptor-binding part of HK620 tailspike and determined its crystal structure to 1.38 {\AA} resolution. Its major domain is a right-handed parallel ;-helix, as in Sf6 and P22 tailspikes. HK620 tailspike has endo-N- acetylglucosaminidase activity and produces hexasaccharides of an O18A1-type O-antigen. As indicated by the structure of a hexasaccharide complex determined at 1.6 {\AA} resolution, the endoglycosidase-active sites are located intramolecularly, as in P22, and not between subunits, as in Sf6 tailspike. In contrast, the extreme C-terminal domain of HK620 tailspike forms a ;-sandwich, as in Sf6 and unlike P22 tailspike. Despite the different folds, structure-based sequence alignments of the C-termini reveal motifs conserved between the three proteins. We propose that the tailspike genes of P22, Sf6 and HK620 have a common precursor and are not mosaics of unrelated gene fragments.},
  language  = {en}
}
@article{BarbirzBeckerFreibergetal.2009,
  author    = {Barbirz, Stefanie and Becker, Marion and Freiberg, Alexander and Seckler, Robert},
  title     = {Phage tailspike proteins with beta-solenoid fold as thermostable carbohydrate binding materials},
  issn      = {1616-5187},
  doi       = {10.1002/mabi.200800278},
  year      = {2009},
  abstract  = {We have investigated the stability of three tailspike proteins (TSPs) from bacteriophages Sf6, P22, and HK620. Tailspikes are rod-like homotrimers with comparable beta-solenoid folds and similarly high kinetic stability in spite of different amino acid sequences. As tailspikes bind polysaccharides to recognize the bacterial host cell, their stability is required for maintenance of bacteriophage infectivity under harsh extracellular conditions. They resist denaturation by SDS at ambient temperature and their unfolding is slow even in 6 m guanidinium hydrochloride (GdmHCl). This makes them interesting candidates for very stable carbohydrate binding protein materials.},
  language  = {en}
}
@article{AndresRoskeDoeringetal.2012,
  author    = {Andres, Dorothee and Roske, Yvette and Doering, Carolin and Heinemann, Udo and Seckler, Robert and Barbirz, Stefanie},
  title     = {Tail morphology controls DNA release in two Salmonella phages with one lipopolysaccharide receptor recognition system},
  series = {Molecular microbiology},
  volume    = {83},
  journal   = {Molecular microbiology},
  number    = {6},
  publisher = {Wiley-Blackwell},
  address   = {Hoboken},
  issn      = {0950-382X},
  doi       = {10.1111/j.1365-2958.2012.08006.x},
  pages     = {1244 -- 1253},
  year      = {2012},
  abstract  = {Bacteriophages use specific tail proteins to recognize host cells. It is still not understood to molecular detail how the signal is transmitted over the tail to initiate infection. We have analysed in vitro DNA ejection in long-tailed siphovirus 9NA and short-tailed podovirus P22 upon incubation with Salmonella typhimurium lipopolysaccharide (LPS). We showed for the first time that LPS alone was sufficient to elicit DNA release from a siphovirus in vitro. Crystal structure analysis revealed that both phages use similar tailspike proteins for LPS recognition. Tailspike proteins hydrolyse LPS O antigen to position the phage on the cell surface. Thus we were able to compare in vitro DNA ejection processes from two phages with different morphologies with the same receptor under identical experimental conditions. Siphovirus 9NA ejected its DNA about 30 times faster than podovirus P22. DNA ejection is under control of the conformational opening of the particle and has a similar activation barrier in 9NA and P22. Our data suggest that tail morphology influences the efficiencies of particle opening given an identical initial receptor interaction event.},
  language  = {en}
}
@article{AndresHankeBaxaetal.2010,
  author    = {Andres, Dorothee and Hanke, Christin and Baxa, Ulrich and Seul, Anait and Barbirz, Stefanie and Seckler, Robert},
  title     = {Tailspike interactions with lipopolysaccharide effect DNA ejection from phage P22 particles in vitro},
  issn      = {0021-9258},
  doi       = {10.1074/jbc.M110.169003},
  year      = {2010},
  abstract  = {Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tail-spikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.},
  language  = {en}
}
@article{AndresGohlkeBroekeretal.2013,
  author    = {Andres, Dorothee and Gohlke, Ulrich and Br{\"o}ker, Nina Kristin and Schulze, Stefan and Rabsch, Wolfgang and Heinemann, Udo and Barbirz, Stefanie and Seckler, Robert},
  title     = {An essential serotype recognition pocket on phage P22 tailspike protein forces Salmonella enterica serovar Paratyphi A O-antigen fragments to bind as nonsolution conformers},
  series = {Glycobiology},
  volume    = {23},
  journal   = {Glycobiology},
  number    = {4},
  publisher = {Oxford Univ. Press},
  address   = {Cary},
  issn      = {0959-6658},
  doi       = {10.1093/glycob/cws224},
  pages     = {486 -- 494},
  year      = {2013},
  abstract  = {Bacteriophage P22 recognizes O-antigen polysaccharides of Salmonella enterica subsp. enterica (S.) with its tailspike protein (TSP). In the serovars S. Typhimurium, S. Enteritidis, and S. Paratyphi A, the tetrasaccharide repeat units of the respective O-antigens consist of an identical main chain trisaccharide but different 3,6-dideoxyhexose substituents. Here, the epimers abequose, tyvelose and paratose determine the specific serotype. P22 TSP recognizes O-antigen octasaccharides in an extended binding site with a single 3,6-dideoxyhexose binding pocket. We have isolated S. Paratyphi A octasaccharides which were not available previously and determined the crystal structure of their complex with P22 TSP. We discuss our data together with crystal structures of complexes with S. Typhimurium and S. Enteritidis octasaccharides determined earlier. Isothermal titration calorimetry showed that S. Paratyphi A octasaccharide binds P22 TSP less tightly, with a difference in binding free energy of similar to 7 kJ mol(-1) at 20 degrees C compared with S. Typhimurium and S. Enteritidis octasaccharides. Individual protein-carbohydrate contacts were probed by amino acid replacements showing that the dideoxyhexose pocket contributes to binding of all three serotypes. However, S. Paratyphi A octasaccharides bind in a conformation with an energetically unfavorable phi/epsilon glycosidic bond angle combination. In contrast, octasaccharides from the other serotypes bind as solution-like conformers. Two water molecules are conserved in all P22 TSP complexes with octasaccharides of different serotypes. They line the dideoxyhexose binding pocket and force the S. Paratyphi A octasaccharides to bind as nonsolution conformers. This emphasizes the role of solvent as part of carbohydrate binding sites.},
  language  = {en}
}
@article{AndresBaxaHankeetal.2010,
  author    = {Andres, Dorothee and Baxa, Ulrich and Hanke, Christin and Seckler, Robert and Barbirz, Stefanie},
  title     = {Carbohydrate binding of Salmonella phage P22 tailspike protein and its role during host cell infection},
  issn      = {0300-5127},
  doi       = {10.1042/Bst0381386},
  year      = {2010},
  abstract  = {TSPs (tailspike proteins) are essential infection organelles of bacteriophage P22. Upon infection, P22TSP binds to and cleaves the O-antigen moiety of the LPS (lipopolysaccharide) of its Salmonella host To elucidate the role of TSP during infection, we have studied binding to oligosaccharides and polysaccharides of Salmonella enteric Typhimurium and Enteritidis in vitro. P22TSP is a trimeric beta-helical protein with a carbohydrate-binding site on each subunit. Octasaccharide O-antigen fragments bind to P22TSP with micromolar dissociation constants. Moreover, P22TSP is an endorhamnosidase and cleaves the host O-antigen. Catalytic residues lie at the periphery of the high-affinity binding site, which enables unproductive binding modes, resulting in slow hydrolysis. However, the role of this hydrolysis function during infection remains unclear. Binding of polysaccharide to P22TSP is of high avidity with slow dissociation rates when compared with oligosaccharides. In vivo, the infection of Salmonella with phage P22 can be completely inhibited by the addition of LPS, indicating that binding of phage to its host via TSP is an essential step for infection.},
  language  = {en}
}