Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation

Ponnuraj, Karthe; Rowland, Susan; Nessi, Claudio; Setlow, Peter; Jedrzejas, Mark J.

doi:10.1006/jmbi.2000.3849

Cited by 20 publications

(11 citation statements)

References 32 publications

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“…This shortage was confirmed by the detection of a lower protein band with an apparent M r of *35.5 kDa. At the same time this result indicates that the lengths of GPR propeptides are not conserved because propeptides of B. megaterium (15 aa) and B. subtilis (16 aa) showed more than twice the size (Ponnuraj et al, 2000;Carroll, 2008) and the first 10 aa could be removed without significant changes of autoprocessing or enzymic activity (Pedersen et al, 1997).…”

Section: Discussionmentioning

confidence: 95%

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Wetzel

Fischer

2015

Microbiology

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Section: Discussionmentioning

confidence: 95%

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Wetzel

Fischer

2015

Microbiology

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“…B. megaterium GPR was chosen for this mutagenesis study because the genes encoding both the P 46 and P 41 forms of this protein have been cloned (28), the purified proteins have proven amenable to in vitro studies (8), and the crystal structure of B. megaterium P 46 has been solved (18). Amino acids in B. megaterium GPR were chosen for change if they fit three criteria.…”

Section: Resultsmentioning

confidence: 99%

“…Figure 5 illustrates the relative orientations of the catalytic aspartic residues of pepsinogen (27) (Fig. 5A) and GPR (18) (Fig. 5B).…”

Section: Resultsmentioning

confidence: 99%

“…Further, K223 appears to interact closely with both putative catalytic aspartic acid residues in a manner reminiscent of pepsinogen's structure (5), and measurement of the distance between C ␦ of D127 and that of D193 (4.4 Å) indicates that they may be held in an inactive conformation. However, this determination is complicated by the low resolution of the P 46 crystal structure (crystals diffracted to 3.0 Å [18]). Finally, the putative inhibitory loop of GPR has a pI significantly higher than that of the protein as a whole (10.0 and 4.9, respectively) (20).…”

Section: Discussionmentioning

confidence: 99%

“…The only detailed structural information on GPR is from the crystal structure of B. megaterium P 46 (18), and consequently the details of structural changes that occur during the P 46 to P 41 conversion are not known. Conversion of P 46 to P 41 is not accompanied by a large change in GPR's secondary structure, as the circular dichroism (CD) spectra of P 46 and P 41 are very similar (11) (see below).…”

mentioning

confidence: 99%

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Site-Directed Mutagenesis and Structural Studies Suggest that the Germination Protease, GPR, in Spores ofBacillusSpecies Is an Atypical Aspartic Acid Protease

Carroll

Setlow

2005

J Bacteriol

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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 (P 41 ) was assayed for activity against SASP and the zymogen form (P 46 ) was assayed for the ability to autoprocess to P 41 . Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multiangle 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 P 46 and P 41 , 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 P 46 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.During the first minutes of germination of spores of Bacillus species, 10 to 20% of the spore's total protein is degraded to amino acids (26). The majority of the degraded protein is a group of small, acid-soluble spore proteins (SASP), some of which (␣/␤ type; SASP-A and -C in Bacillus megaterium) bind to the spore's DNA and contribute to its resistance against heat and UV radiation (14,22,23,26). Another SASP subtype (␥ type; SASP-B in B. megaterium) does not bind spore DNA but rather, along with the ␣/␤ type SASP, serves as an important amino acid reserve in the mature spore (4). Germination protease (GPR) initiates SASP degradation by cleaving these proteins at one or two sites, depending on the SASP subtype, thereby freeing the spore's DNA and ultimately providing amino acids for metabolism and protein synthesis in spore outgrowth (26).GPR is a sequence-specific endoprotease that is synthesized as an inactive zymogen (P 46 ) during sporulation at about the same time as its SASP substrates (26). Just prior to the completion of sporulation, GPR undergoes an autoprocessing reaction which removes an N-terminal propeptide of 5 to 15 amino acids, depending on the species (13, 21). This process results in an enzyme (P 41 ) that is active in vitro but does not cleave SASP in vivo due to the relatively low water content in the core of the developing and dormant spore (7,13,21). In vitro, autoprocessing is triggered by pyridine-2,6-di...

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Structural insights into the psychrophilic germinal protease PaGPR and its autoinhibitory loop

Lee

Jeong

et al. 2020

J Microbiol.

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Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation

Cited by 20 publications

References 32 publications

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Site-Directed Mutagenesis and Structural Studies Suggest that the Germination Protease, GPR, in Spores ofBacillusSpecies Is an Atypical Aspartic Acid Protease

Structural insights into the psychrophilic germinal protease PaGPR and its autoinhibitory loop

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