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PDBsum entry 1a2q
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Hydrolase PDB id
1a2q

 

 

 

 

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Contents
Protein chain
275 a.a. *
Ligands
ACN ×2
Metals
_CA ×2
Waters ×186
* Residue conservation analysis
PDB id:
1a2q
Name: Hydrolase
Title: Subtilisin bpn' mutant 7186
Structure: Subtilisin bpn'. Chain: a. Engineered: yes. Mutation: yes
Source: Bacillus amyloliquefaciens. Organism_taxid: 1390. Strain: gx7186. Cellular_location: secreted. Expressed in: bacillus subtilis. Expression_system_taxid: 1423.
Resolution:
1.80Å     R-factor:   0.145    
Authors: G.L.Gilliland,M.Whitlow,A.J.Howard
Key ref:
M.W.Pantoliano et al. (1989). Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding. Biochemistry, 28, 7205-7213. PubMed id: 2684274 DOI: 10.1021/bi00444a012
Date:
08-Jan-98     Release date:   29-Apr-98    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
P00782  (SUBT_BACAM) -  Subtilisin BPN' from Bacillus amyloliquefaciens
Seq:
Struc: 		382 a.a.
275 a.a.*
Key:    Secondary structure  CATH domain
* PDB and UniProt seqs differ at 5 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.3.4.21.62  - subtilisin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.

 

 
DOI no: 10.1021/bi00444a012 Biochemistry 28:7205-7213 (1989)
PubMed id: 2684274  
 
 
Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding.
M.W.Pantoliano, M.Whitlow, J.F.Wood, S.W.Dodd, K.D.Hardman, M.L.Rollence, P.N.Bryan.
 
  ABSTRACT  
 
Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
20519323 A.Badoei-Dalfard, K.Khajeh, S.M.Asghari, B.Ranjbar, and H.R.Karbalaei-Heidari (2010).
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19763902 O.Prakash, and N.Jaiswal (2010).
alpha-Amylase: an ideal representative of thermostable enzymes.
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20100702 T.Foophow, S.Tanaka, Y.Koga, K.Takano, and S.Kanaya (2010).
Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides.
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20095051 W.A.Baase, L.Liu, D.E.Tronrud, and B.W.Matthews (2010).
Lessons from the lysozyme of phage T4.
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19805099 C.Ottmann, R.Rose, F.Huttenlocher, A.Cedzich, P.Hauske, M.Kaiser, R.Huber, and A.Schaller (2009).
Structural basis for Ca2+-independence and activation by homodimerization of tomato subtilase 3.
  Proc Natl Acad Sci U S A, 106, 17223-17228.
PDB codes: 3i6s 3i74
19381264 J.D.Bloom, and M.J.Glassman (2009).
Inferring stabilizing mutations from protein phylogenies: application to influenza hemagglutinin.
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22477769 R.P.Joosten, J.Salzemann, V.Bloch, H.Stockinger, A.C.Berglund, C.Blanchet, E.Bongcam-Rudloff, C.Combet, A.L.Da Costa, G.Deleage, M.Diarena, R.Fabbretti, G.Fettahi, V.Flegel, A.Gisel, V.Kasam, T.Kervinen, E.Korpelainen, K.Mattila, M.Pagni, M.Reichstadt, V.Breton, I.J.Tickle, and G.Vriend (2009).
PDB_REDO: automated re-refinement of X-ray structure models in the PDB.
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18626642 Y.H.Liu, F.P.Lu, Y.Li, J.L.Wang, and C.Gao (2008).
Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations.
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17944939 M.Haruki, M.Tanaka, T.Motegi, T.Tadokoro, Y.Koga, K.Takano, and S.Kanaya (2007).
Structural and thermodynamic analyses of Escherichia coli RNase HI variant with quintuple thermostabilizing mutations.
  FEBS J, 274, 5815-5825.
PDB code: 2yv0
17237225 S.Tanaka, K.Saito, H.Chon, H.Matsumura, Y.Koga, K.Takano, and S.Kanaya (2007).
Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding.
  J Biol Chem, 282, 8246-8255.
PDB code: 2e1p
16751527 M.Pulido, K.Saito, S.Tanaka, Y.Koga, M.Morikawa, K.Takano, and S.Kanaya (2006).
Ca2+-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.
  Appl Environ Microbiol, 72, 4154-4162.  
15618218 A.I.Solbak, T.H.Richardson, R.T.McCann, K.A.Kline, F.Bartnek, G.Tomlinson, X.Tan, L.Parra-Gessert, G.J.Frey, M.Podar, P.Luginbühl, K.A.Gray, E.J.Mathur, D.E.Robertson, M.J.Burk, G.P.Hazlewood, J.M.Short, and J.Kerovuo (2005).
Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric.
  J Biol Chem, 280, 9431-9438.  
15128565 J.B.Garrett, K.A.Kretz, E.O'Donoghue, J.Kerovuo, W.Kim, N.R.Barton, G.P.Hazlewood, J.M.Short, D.E.Robertson, and K.A.Gray (2004).
Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement.
  Appl Environ Microbiol, 70, 3041-3046.  
15298890 L.Yang, J.S.Dordick, and S.Garde (2004).
Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity.
  Biophys J, 87, 812-821.  
12649438 R.M.Day, C.J.Thalhauser, J.L.Sudmeier, M.P.Vincent, E.V.Torchilin, D.G.Sanford, C.W.Bachovchin, and W.W.Bachovchin (2003).
Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN' by NMR.
  Protein Sci, 12, 794-810.  
11551465 M.Lehmann, and M.Wyss (2001).
Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution.
  Curr Opin Biotechnol, 12, 371-375.  
11606307 S.Lee, and D.J.Jang (2001).
Progressive rearrangement of subtilisin Carlsberg into orderly and inflexible conformation with Ca(2+) binding.
  Biophys J, 81, 2972-2978.  
11150607 P.N.Bryan (2000).
Protein engineering of subtilisin.
  Biochim Biophys Acta, 1543, 203-222.  
11023921 S.Lee, and D.J.Jang (2000).
Cation-binding sites of subtilisin Carlsberg probed with Eu(III) luminescence.
  Biophys J, 79, 2171-2177.  
10194383 G.Feller, D.d'Amico, and C.Gerday (1999).
Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis.
  Biochemistry, 38, 4613-4619.  
10097079 P.J.Haney, J.H.Badger, G.L.Buldak, C.I.Reich, C.R.Woese, G.J.Olsen, and C.R.Woese (1999).
Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species.
  Proc Natl Acad Sci U S A, 96, 3578-3583.  
  10048334 R.D.Kidd, P.Sears, D.H.Huang, K.Witte, C.H.Wong, and G.K.Farber (1999).
Breaking the low barrier hydrogen bond in a serine protease.
  Protein Sci, 8, 410-417.
PDB codes: 1yja 1yjb 1yjc
10095787 S.Akanuma, A.Yamagishi, N.Tanaka, and T.Oshima (1999).
Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3-isopropylmalate dehydrogenase variant by random and site-directed mutagenesis.
  Eur J Biochem, 260, 499-504.  
9482832 F.H.Arnold (1998).
Enzyme engineering reaches the boiling point.
  Proc Natl Acad Sci U S A, 95, 2035-2036.  
  9541409 L.A.Lipscomb, N.C.Gassner, S.D.Snow, A.M.Eldridge, W.A.Baase, D.L.Drew, and B.W.Matthews (1998).
Context-dependent protein stabilization by methionine-to-leucine substitution shown in T4 lysozyme.
  Protein Sci, 7, 765-773.
PDB codes: 230l 231l 232l 233l 234l
9552156 O.Almog, T.Gallagher, M.Tordova, J.Hoskins, P.Bryan, and G.L.Gilliland (1998).
Crystal structure of calcium-independent subtilisin BPN' with restored thermal stability folded without the prodomain.
  Proteins, 31, 21-32.
PDB code: 1sua
9843948 P.V.Nikolova, J.Henckel, D.P.Lane, and A.R.Fersht (1998).
Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability.
  Proc Natl Acad Sci U S A, 95, 14675-14680.  
  9541402 S.Akanuma, A.Yamagishi, N.Tanaka, and T.Oshima (1998).
Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.
  Protein Sci, 7, 698-705.  
9628485 S.M.Malakauskas, and S.L.Mayo (1998).
Design, structure and stability of a hyperthermophilic protein variant.
  Nat Struct Biol, 5, 470-475.
PDB code: 1gb4
9484240 V.De Filippis, F.De Antoni, M.Frigo, P.Polverino de Laureto, and A.Fontana (1998).
Enhanced protein thermostability by Ala-->Aib replacement.
  Biochemistry, 37, 1686-1696.  
  9007995 E.C.Ohage, W.Graml, M.M.Walter, S.Steinbacher, and B.Steipe (1997).
Beta-turn propensities as paradigms for the analysis of structural motifs to engineer protein stability.
  Protein Sci, 6, 233-241.  
9115441 J.R.Martin, F.A.Mulder, Y.Karimi-Nejad, J.van der Zwan, M.Mariani, D.Schipper, and R.Boelens (1997).
The solution structure of serine protease PB92 from Bacillus alcalophilus presents a rigid fold with a flexible substrate-binding site.
  Structure, 5, 521-532.
PDB code: 1ah2
9188450 M.A.Kemper, C.D.Stout, S.J.Lloyd, G.S.Prasad, S.E.Fawcett, F.A.Armstrong, B.Shen, B.K.Burgess, S.E.Lloyd, and S.Fawcett (1997).
Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.
  J Biol Chem, 272, 15620-15627.
PDB code: 1ftc
9048549 T.Vogl, C.Jatzke, H.J.Hinz, J.Benz, and R.Huber (1997).
Thermodynamic stability of annexin V E17G: equilibrium parameters from an irreversible unfolding reaction.
  Biochemistry, 36, 1657-1668.  
8738345 A.Sättler, S.Kanka, K.H.Maurer, and D.Riesner (1996).
Thermostable variants of subtilisin selected by temperature-gradient gel electrophoresis.
  Electrophoresis, 17, 784-792.  
  8528073 J.J.Onuffer, and J.F.Kirsch (1995).
Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.
  Protein Sci, 4, 1750-1757.  
7499319 O.E.Janssen, B.Chen, C.Büttner, S.Refetoff, and P.C.Scriba (1995).
Molecular and structural characterization of the heat-resistant thyroxine-binding globulin-Chicago.
  J Biol Chem, 270, 28234-28238.  
8556430 P.W.Goodenough (1995).
A review of protein engineering for the food industry.
  Mol Biotechnol, 4, 151-166.  
9634803 S.L.Strausberg, P.A.Alexander, D.T.Gallagher, G.L.Gilliland, B.L.Barnett, and P.N.Bryan (1995).
Directed evolution of a subtilisin with calcium-independent stability.
  Biotechnology (N Y), 13, 669-673.  
8535784 T.Gallagher, G.Gilliland, L.Wang, and P.Bryan (1995).
The prosegment-subtilisin BPN' complex: crystal structure of a specific 'foldase'.
  Structure, 3, 907-914.
PDB code: 1spb
8033905 A.Gase, E.Birch-Hirschfeld, K.H.Gührs, M.Hartmann, S.Vetterman, G.Damaschun, H.Damaschun, K.Gast, R.Misselwitz, and D.Zirwer (1994).
The thermostability of natural variants of bacterial plasminogen-activator staphylokinase.
  Eur J Biochem, 223, 303-308.  
  7987227 P.Völkl, P.Markiewicz, K.O.Stetter, and J.H.Miller (1994).
The sequence of a subtilisin-type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability.
  Protein Sci, 3, 1329-1340.  
7809074 T.K.Chang, D.Y.Jackson, J.P.Burnier, and J.A.Wells (1994).
Subtiligase: a tool for semisynthesis of proteins.
  Proc Natl Acad Sci U S A, 91, 12544-12548.  
8451240 C.X.Wang, Y.Y.Shi, F.Zhou, and L.Wang (1993).
Thermodynamic integration calculations of binding free energy difference for Gly-169 mutation in subtilisin BPN'.
  Proteins, 15, 5-9.  
8229092 G.Vriend, and V.Eijsink (1993).
Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases.
  J Comput Aided Mol Des, 7, 367-396.  
8508784 I.G.Sumner, G.W.Harris, M.A.Taylor, R.W.Pickersgill, A.J.Owen, and P.W.Goodenough (1993).
Factors effecting the thermostability of cysteine proteinases from Carica papaya.
  Eur J Biochem, 214, 129-134.  
8516309 K.Chen, and F.H.Arnold (1993).
Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.
  Proc Natl Acad Sci U S A, 90, 5618-5622.  
8332608 T.Gallagher, P.Bryan, and G.L.Gilliland (1993).
Calcium-independent subtilisin by design.
  Proteins, 16, 205-213.
PDB codes: 1sub 1suc 1sud
8378307 W.S.Sandberg, and T.C.Terwilliger (1993).
Engineering multiple properties of a protein by combinatorial mutagenesis.
  Proc Natl Acad Sci U S A, 90, 8367-8371.  
1553381 P.Gros, A.V.Teplyakov, and W.G.Hol (1992).
Effects of eglin-c binding to thermitase: three-dimensional structure comparison of native thermitase and thermitase eglin-c complexes.
  Proteins, 12, 63-74.  
18600924 P.Martinez, M.E.Van Dam, A.C.Robinson, K.Chen, and F.H.Arnold (1992).
Stabilization of substilisin E in organic solvents by site-directed mutagenesis.
  Biotechnol Bioeng, 39, 141-147.  
1476392 T.P.Graycar, R.R.Bott, R.M.Caldwell, J.L.Dauberman, P.J.Lad, S.D.Power, I.H.Sagar, R.A.Silva, G.L.Weiss, and L.R.Woodhouse (1992).
Altering the proteolytic activity of subtilisin through protein engineering.
  Ann N Y Acad Sci, 672, 71-79.  
1915340 F.Conejero-Lara, J.M.Sánchez-Ruiz, P.L.Mateo, F.J.Burgos, J.Vendrell, and F.X.Avilés (1991).
Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain.
  Eur J Biochem, 200, 663-670.  
1366953 W.S.Sandberg, and T.C.Terwilliger (1991).
Repacking protein interiors.
  Trends Biotechnol, 9, 59-63.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.

 

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