PDBsum entry 1ob0

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protein metals links
Hydrolase PDB id
Protein chain
481 a.a. *
_CA ×3
Waters ×324
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Kinetic stabilization of bacillus licheniformis alpha-amylas through introduction of hydrophobic residues at the surface
Structure: Alpha-amylase. Chain: a. Synonym: 1,4-alpha-d-glucan-4-glucanohydrolase. Engineered: yes. Mutation: yes
Source: Bacillus licheniformis. Organism_taxid: 1402. Expressed in: bacillus subtilis. Expression_system_taxid: 1423
1.83Å     R-factor:   0.147     R-free:   0.154
Authors: M.Machius,N.Declerck,R.Huber,G.Wiegand
Key ref:
M.Machius et al. (2003). Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface. J Biol Chem, 278, 11546-11553. PubMed id: 12540849 DOI: 10.1074/jbc.M212618200
21-Jan-03     Release date:   30-Jan-03    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P06278  (AMY_BACLI) -  Alpha-amylase
512 a.a.
481 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 8 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Alpha-amylase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     metabolic process   2 terms 
  Biochemical function     catalytic activity     8 terms  


DOI no: 10.1074/jbc.M212618200 J Biol Chem 278:11546-11553 (2003)
PubMed id: 12540849  
Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface.
M.Machius, N.Declerck, R.Huber, G.Wiegand.
It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.
  Selected figure(s)  
Figure 2.
Fig. 2. Crystal structure of kinetically stabilized BLA. A, representative 2F[o] F[c] simulated annealing omit electron density map for a region in the core of the kinetically stabilized BLA variant. B, stereoview of a schematic representation of the overall structure of BLA with the mutation sites labeled. Calcium ions are shown in cyan, and sodium is in yellow. All figures were created using the programs Bobscript (54) and POVRray (Persistence of Vision, v3.02, POV-Team, and GL_RENDER (L. Esser, University of Texas Southwestern Medical Center).
Figure 4.
Fig. 4. Stereoview of the region around the Ca-Na-Ca metal triad containing the mutation N190F. Calcium ions are shown in cyan and the sodium ion is in yellow; other atoms are in standard colors.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 11546-11553) copyright 2003.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21190177 B.Pierre, T.Xiong, L.Hayles, V.R.Guntaka, and J.R.Kim (2011).
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Modulation of contact order effects in the two-state folding of stefins a and B.
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The critical role of N- and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions.
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20572789 C.Neeraja, K.Anil, P.Purushotham, K.Suma, P.Sarma, B.M.Moerschbacher, and A.R.Podile (2010).
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Structural insights into the specificity of Xyn10B from Paenibacillus barcinonensis and its improved stability by forced protein evolution.
  J Biol Chem, 285, 2721-2733.
PDB codes: 3emc 3emq 3emz
19763902 O.Prakash, and N.Jaiswal (2010).
alpha-Amylase: an ideal representative of thermostable enzymes.
  Appl Biochem Biotechnol, 160, 2401-2414.  
20528916 S.Kumar, N.Singh, M.Sinha, D.Dube, S.B.Singh, A.Bhushan, P.Kaur, A.Srinivasan, S.Sharma, and T.P.Singh (2010).
Crystal structure determination and inhibition studies of a novel xylanase and alpha-amylase inhibitor protein (XAIP) from Scadoxus multiflorus.
  FEBS J, 277, 2868-2882.
PDB codes: 3hu7 3m7s
19201981 I.A.Rasiah, and B.H.Rehm (2009).
One-step production of immobilized alpha-amylase in recombinant Escherichia coli.
  Appl Environ Microbiol, 75, 2012-2016.  
19504261 S.C.Yadav, and M.V.Jagannadham (2009).
Complete conformational stability of kinetically stable dimeric serine protease milin against pH, temperature, urea, and proteolysis.
  Eur Biophys J, 38, 981-991.  
18725971 A.Bharadwaj, S.Leelavathi, S.Mazumdar-Leighton, A.Ghosh, S.Ramakumar, and V.S.Reddy (2008).
The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp.NG-27 under poly-extreme conditions.
  PLoS ONE, 3, e3063.  
18451358 B.F.Shaw, G.F.Schneider, B.Bilgiçer, G.K.Kaufman, J.M.Neveu, W.S.Lane, J.P.Whitelegge, and G.M.Whitesides (2008).
Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation.
  Protein Sci, 17, 1446-1455.  
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.
  Appl Microbiol Biotechnol, 80, 795-803.  
18157528 Y.H.Liu, F.P.Lu, Y.Li, X.B.Yin, Y.Wang, and C.Gao (2008).
Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600.
  Appl Microbiol Biotechnol, 78, 85-94.  
15688447 H.K.Liang, C.M.Huang, M.T.Ko, and J.K.Hwang (2005).
Amino acid coupling patterns in thermophilic proteins.
  Proteins, 59, 58-63.  
15803194 S.Frokjaer, and D.E.Otzen (2005).
Protein drug stability: a formulation challenge.
  Nat Rev Drug Discov, 4, 298-306.  
15857780 V.G.Eijsink, S.Gåseidnes, T.V.Borchert, and B.van den Burg (2005).
Directed evolution of enzyme stability.
  Biomol Eng, 22, 21-30.  
15819891 Y.S.Yun, G.H.Nam, Y.G.Kim, B.H.Oh, and K.Y.Choi (2005).
Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B.
  FEBS J, 272, 1999-2011.
PDB code: 1w6y
14760745 A.Linden, and M.Wilmanns (2004).
Adaptation of class-13 alpha-amylases to diverse living conditions.
  Chembiochem, 5, 231-239.  
15532068 C.H.Chan, H.K.Liang, N.W.Hsiao, M.T.Ko, P.C.Lyu, and J.K.Hwang (2004).
Relationship between local structural entropy and protein thermostability.
  Proteins, 57, 684-691.  
15457404 J.Chen, B.K.Lipska, N.Halim, Q.D.Ma, M.Matsumoto, S.Melhem, B.S.Kolachana, T.M.Hyde, M.M.Herman, J.Apud, M.F.Egan, J.E.Kleinman, and D.R.Weinberger (2004).
Functional analysis of genetic variation in catechol-O-methyltransferase (COMT): effects on mRNA, protein, and enzyme activity in postmortem human brain.
  Am J Hum Genet, 75, 807-821.  
15479244 J.Köditz, R.Ulbrich-Hofmann, and U.Arnold (2004).
Probing the unfolding region of ribonuclease A by site-directed mutagenesis.
  Eur J Biochem, 271, 4147-4156.  
14718652 N.Palackal, Y.Brennan, W.N.Callen, P.Dupree, G.Frey, F.Goubet, G.P.Hazlewood, S.Healey, Y.E.Kang, K.A.Kretz, E.Lee, X.Tan, G.L.Tomlinson, J.Verruto, V.W.Wong, E.J.Mathur, J.M.Short, D.E.Robertson, and B.A.Steer (2004).
An evolutionary route to xylanase process fitness.
  Protein Sci, 13, 494-503.  
12943855 J.R.Cherry, and A.L.Fidantsef (2003).
Directed evolution of industrial enzymes: an update.
  Curr Opin Biotechnol, 14, 438-443.  
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.