The 0.93Å Crystal Structure of Sphericase: A Calcium-loaded Serine Protease from Bacillus sphaericus

Almog, Orna; González, Ana; Klein, D.; Greenblatt, H.M.; Braun, Sergei; Shoham, G.

doi:10.1016/j.jmb.2003.07.011

Cited by 39 publications

(43 citation statements)

References 55 publications

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“…In the case of sphericase; a serine protease from Bacillus sphaericus, up to five calcium ions have been found associated with a single enzyme molecule (Almog et al 2003), where as in the case of a marine Vibrio proteinase, three Ca ions were found associated with its structure, one of which was in a calcium binding site not described in other subtilases (Arnorsdottir et al 2005). Amongst the three additional calcium binding sites predicted in EAP, the one which is formed by residues Ala11, Ser14, Ser14, Ser22 is similar to a calcium binding site reported in the marine Vibrio proteinase, which is formed by residues Asp11, Asp14, Gln15, Asp21 and Asn23(PRK1 numbering) (Arnorsdottir et al 2005).…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

Corso

Chellappan

Sukumaran

et al. 2010

World J Microbiol Biotechnol

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An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca 2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.Keywords Engyodontium album Á Alkaline serine protease Á Subtilases Á Homology modelling Abbreviations E. album Engyodontium album EAPEngyodontium album protease (deduced protein) Eap Engyodontium album protease (gene)

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Section: Discussionmentioning

confidence: 99%

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

Corso

Chellappan

Sukumaran

et al. 2010

World J Microbiol Biotechnol

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“…For example, two binding sites were found for the mesophilic subtilisin BPN' (pdb entry 1sup), three were found for the thermophilic subtilisin thermitase 67 (pdb entry 1thm) and the subtilisin-like serine protease from the psychrophilic Vibrio species (pdb entry 1sh7) 68 . Five sites were found for the mesophilic subtilisin Sph (pdb entry 1ea7) 69 and the psychrophilic subtilisin S41…”

Section: Cold Adaptationmentioning

confidence: 99%

Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41

et al. 2007

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A wide variety of enzymes can undergo a reversible loss of activity at low temperature, a process that is termed cold inactivation. This phenomenon is found in oligomeric enzymes such as tryptophanase (Trpase) and other pyridoxal phosphate dependent enzymes. On the other hand, cold‐adapted, or psychrophilic enzymes, isolated from organisms able to thrive in permanently cold environments, have optimal activity at low temperature, which is associated with low thermal stability. Since cold inactivation may be considered “contradictory” to cold adaptation, we have looked into the amino acid sequences and the crystal structures of two families of enzymes, subtilisin and tryptophanase. Two cold adapted subtilisins, S41 and subtilisin‐like protease from Vibrio, were compared to a mesophilic and a thermophilic subtilisins, as well as to four PLP‐dependent enzymes in order to understand the specific surface residues, specific interactions, or any other molecular features that may be responsible for the differences in their tolerance to cold temperatures. The comparison between the psychrophilic and the mesophilic subtilisins revealed that the cold adapted subtilisins have a high content of acidic residues mainly found on their surface, making it charged. The analysis of the Trpases showed that they have a high content of hydrophobic residues on their surface. Thus, we suggest that the negatively charged residues on the surface of the subtilisins may be responsible for their cold adaptation, whereas the hydrophobic residues on the surface of monomeric Trpase molecules are responsible for the tetrameric assembly, and may account for their cold inactivation and dissociation. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 354–359, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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“…The subtilisin family includes subtilisins from (hyper)thermophiles (13,27,28,35) and psychrophiles (3,14,29,37). The crystal structures of some of them have been determined (2,4,50,57). These thermostable and thermolabile subtilisins have been regarded not only as good models for studying stability-activitystructure relationships of proteins, but also as potential candidates for various biotechnological applications.…”

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Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Pulido

Saito

Tanaka

et al. 2006

Appl Environ Microbiol

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Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca 2؉ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca 2؉ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin‫)ء‬ but forms an inactive complex with T. kodakaraensis mat-subtilisin‫,ء‬ at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin‫,ء‬ and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca 2؉ but was activated upon incubation with Ca 2؉ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin‫,ء‬ indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ϳ5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin‫ء‬ and noncompetitively inhibited its activity with a K i of 25 ؎ 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.Subtilisin-like serine proteases (subtilases) are widely distributed in various organisms, including bacteria, archaea, and eucaryotes (48). They are divided into six families (47). Of these, the structures and functions of the subtilisin family (EC 3.4.21.108), which is represented by subtilisin E from Bacillus subtilis (52), subtilisin BPNЈ from Bacillus amyloliquefaciens (61), and subtilisin Carlsberg from Bacillus licheniformis (25), have been most extensively studied. The crystal structures of these subtilisins have been determined (26,54,64). Because subtilisins are commercially valuable enzymes, extensive attempts to improve their activities and stabilities with proteinengineering technology have also been made (10, 55, 60). The subtilisin family includes subtilisins from (hyper)thermophiles (13,27,28,35) and psychrophiles (3,14,29,37). The crystal structures of some of them have been determined (2,4,50,57). These thermostable and thermolabile subtilisins have been regarded not only as good models for studying stability-activitystructure relationships of proteins, but also as potent...

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The 0.93Å Crystal Structure of Sphericase: A Calcium-loaded Serine Protease from Bacillus sphaericus

Cited by 39 publications

References 55 publications

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41

Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

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The 0.93Å Crystal Structure of Sphericase: A Calcium-loaded Serine Protease from Bacillus sphaericus

Cited by 39 publications

References 55 publications

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41

Ca 2+ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

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Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding