In response to environmental stress Bacillus and Clostridium species undergo a complex adaptive response called sporulation. This process results in the formation of the endospore, a metabolically quiescent and highly resistant structure that allows these species to survive conditions that kill other microorganisms. While carrying out no detectible metabolic activity, the endospore is able to sense the presence of nutrients sufficient for vegetative survival and in response undergoes the process of germination which converts the dormant endospore into a vegetatively growing and dividing cell. Near the end of sporulation two important classes of proteins are synthesized: DNA-protective proteins termed small, acid-soluble spore proteins (SASP), and the zymogen of the germination protease (GPR). SASP bind the spore's DNA and protect it from UV radiation in addition to serving as an amino acid depot that is utilized during spore germination. While the DNA-protective function of SASP is essential for spore viability, their persistence is detrimental to the germinating spore. GPR functions to initiate the degradation of SASP early in spore outgrowth by hydrolyzing one or two peptide bonds, depending on the SASP subtype. GPR is a protease of unknown classification and mechanism of action. It is synthesized late in sporulation as a zymogen. Just prior to the completion of sporulation it undergoes an autoprocessing reaction, yielding the active form of the protease that is prohibited from acting on SASP due to the relatively dehydrated state of the endospore's core. During germination the influx of water rehydrates the endospore's core, allowing GPR to initiate the degradation of SASP. GPR has no significant sequence or three-dimensional similarity to any known protein and has frustrated all previous efforts to identify its catalytic residues. This work utilizes site-directed mutagenesis, enzymatic assays and spectroscopy to identify two aspartates (D127 and D193) as GPR's catalytic residues, thereby supporting the conclusion that GPR is an atypical aspartic acid protease. Further, mapping of these two aspartates onto the crystal structure of GPR's zymogen reveals that their relative position in space is compatible with the catalytic mechanism of an aspartic acid protease.
