Structural studies of a novel germination protease from spores of Bacillus megaterium.
The amino acid sequence-specific protease (termed GPR) in the bacterium Bacillus megaterium initiates the rapid degradation of small, acid-soluble spore proteins during the germination of spores of this organism. GPR is synthesized during spore formation as an inactive zymogen termed P46, which later autoprocesses to a smaller active form termed P41, which acts during spore germination. However, GPR exhibits no obvious mechanistic or amino acid sequence similarity to any of the known classes of proteases. To initiate the determination of the mechanisms of P46 to P41 conversion, P46 inactivity, and P41 catalysis, B. megaterium GPR has been overexpressed in Escherichia coli and purified to homogeneity by anion-exchange and size exclusion chromatography, and crystals of both P46 and P41 have been obtained by the vapor diffusion method. P46 crystals diffracted x rays to 3.5 A but the crystals of P41 diffracted x rays to only 6.5 A. A native x-ray diffraction data set of P46 has been collected; the unit cell parameters are a = b = 76.8, c = 313.1 A, alpha = beta = gamma = 90 degrees; the space group is tetragonal P41212 or P43212. The asymmetric unit contains two monomeric molecules with a crystal volume per unit protein mass of 2. 85 A3/Da and a solvent content of about 57%. An isomorphous heavy atom derivative data set has also been obtained for P46 crystals with potassium dicyanoaurate (I).
Ponnuraj
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Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Nessi
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Setlow
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Jedrzejas
M J
MJ
D001410:Bacillus megaterium / Q000201:enzymology / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry*; D004792:Enzyme Precursors / Q000737:chemistry; D011092:Polyethylene Glycols / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012098:Reproduction; D013170:Spores / Q000201:enzymology; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D011092:Polyethylene Glycols; D010450:Endopeptidases