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1.  Influence of temperature during crystallization setup on precipitate formation and crystal shape of a metalloendopeptidase 
The effect of temperature during crystallization setup was analyzed and was found to influence precipitate formation and the crystal shape of the inactive E294A mutant of A. salmonicida ssp. achromogenes protease 1.
It is well known that protein crystallization is affected by several different parameters such as the composition of the reservoir solution, the protein concentration, the pH and the temperature. An effect of different temperatures during setup of crystallization experiments was observed for a metalloendopeptidase (AsaP1E294A). Spontaneous protein precipitation was reduced and the crystal shape could be improved by decreasing the temperature during crystallization setup.
PMCID: PMC3053177  PMID: 21393857
crystal growth; temperature; precipitation; metalloendopeptidases; AsaP1
2.  Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-I 
The crystallization and preliminary X-ray diffraction studies of DppA from P. pacifica SIR-I are reported.
DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P21212. The DppA crystal diffracted X-rays to 1.9 Å resolution using an in-house X-ray generator.
PMCID: PMC2898472  PMID: 20606284
haloalkane dehalogenases; Plesiocystis pacifica SIR-I
3.  Crystallization and preliminary X-ray crystallographic analysis of the tetracycline-degrading monooxygenase TetX2 from Bacteroides thetaiotaomicron  
Crystallization and preliminary X-ray crystallographic analysis of the tetracycline-degrading monooxygenase TetX2 from B. thetaiotaomicron are reported.
The flavin-dependent monooxygenase TetX2 from Bacteroides thetaiotaomicron confers resistance against tetracyclines in aerobically grown Escherichia coli. TetX2 modifies several tetracycline antibiotics by regioselective hydroxylation of the substrates to 11a-hydroxy-tetracyclines. X-ray diffraction data were collected from a native TetX2 crystal and a TetX2 crystal with incorporated selenomethionine to resolutions of 2.5 and 3.0 Å, respectively. The native crystal belonged to the triclinic space group P1, with unit-cell parameters a = 68.55, b = 80.88, c = 87.53 Å, α = 111.09, β = 98.98, γ = 93.38°, whereas the selenomethionine-labelled TetX2 crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 87.34, b = 68.66, c = 152.48 Å, β = 101.08°.
PMCID: PMC2864705  PMID: 20445272
tetracycline resistance; tetracycline degradation; flavin-dependent monooxygenases; Bacteroides thetaiotaomicron
4.  Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR 
Nucleic Acids Research  2012;40(9):4178-4192.
Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pKa of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14–Cys49′ intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8–9 Å apart from Cys49′. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4′ helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.
PMCID: PMC3351151  PMID: 22238377
5.  Crystallization and preliminary X-ray diffraction studies of AsaP1_E294A and AsaP1_E294Q, two inactive mutants of the toxic zinc metallopeptidase AsaP1 from Aeromonas salmonicida subsp. achromogenes  
Crystallization and preliminary X-ray diffraction studies of AsaP1_E294A and AsaP1_E294Q, two inactive mutants of the toxic zinc metallopeptidase AsaP1 from A. salmonicida subsp. achromogenes, are reported.
Two mutants of the toxic extracellular zinc endopeptidase AsaP1 (AsaP1_E294Q and AsaP1_E294A) of Aeromonas salmonicida subsp. achromogenes were expressed in Escherichia coli and crystallized by the vapour-diffusion method. Crystals were obtained using several precipitants and different protein concentrations. Protein crystals were found in a monoclinic (C2) as well as an orthorhombic (P212121) space group. The crystals belonging to the monoclinic space group C2 had unit-cell parameters a = 103.4, b = 70.9, c = 54.9 Å, β = 109.3° for AsaP1_E294A, and a = 98.5, b = 74.5, c = 54.7 Å, β = 112.4° for AsaP1_E294Q. The unit-cell parameters of the orthorhombic crystal obtained for AsaP1_E294A were a = 57.9, b = 60.2, c = 183.6 Å. The crystals of the two different mutants diffracted X-rays beyond 2.0 Å resolution.
PMCID: PMC2705637  PMID: 19574642
zinc metallopeptidases; Aeromonas salmonicida subsp. achromogenes
6.  Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain 
Crystal structure analysis of the apo form of catabolite control protein A reveals the three-helix bundle of the DNA-binding domain. In the crystal packing, this domain interacts with the binding site for the corepressor protein.
Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer.
PMCID: PMC2330204  PMID: 17401189
catabolite control protein A; carbon catabolite repression; Bacillus megaterium

Results 1-6 (6)