Site-directed mutagenesis and structural studies suggest that the germination protease, GPR, in spores of Bacillus species is an atypical aspartic acid protease.
Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multi-angle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease.
Carroll
Thomas M
TM
Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06032, USA.
Setlow
Peter
P
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism*; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013171:Spores, Bacterial / Q000201:enzymology / Q000235:genetics
D001224:Aspartic Acid; D010450:Endopeptidases; C104642:spore germination protease