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PDBsum entry 1bsi

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protein ligands metals links
Hydrolase PDB id
1bsi
Jmol
Contents
Protein chain
496 a.a. *
Ligands
NAG
Metals
_CL
_CA
Waters ×222
* Residue conservation analysis
PDB id:
1bsi
Name: Hydrolase
Title: Human pancreatic alpha-amylase from pichia pastoris, glycosy protein
Structure: Alpha-amylase. Chain: a. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Organ: pancreas. Expressed in: pichia pastoris. Expression_system_taxid: 4922
Resolution:
2.00Å     R-factor:   0.184    
Authors: E.H.Rydberg,G.Sidhu,H.C.Vo,J.Hewitt,H.C.F.Cote,Y.Wang,S.Numa R.T.A.Macgillivray,C.M.Overall,G.D.Brayer,S.G.Withers
Key ref: E.H.Rydberg et al. (1999). Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris. Protein Sci, 8, 635-643. PubMed id: 10091666 DOI: 10.1110/ps.8.3.635
Date:
28-Aug-98     Release date:   18-May-99    
PROCHECK
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 Headers
 References

Protein chain
Pfam   ArchSchema ?
P04746  (AMYP_HUMAN) -  Pancreatic alpha-amylase
Seq:
Struc:
511 a.a.
496 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.3.2.1.1  - 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!
  Cellular component     extracellular region   3 terms 
  Biological process     metabolic process   5 terms 
  Biochemical function     catalytic activity     8 terms  

 

 
DOI no: 10.1110/ps.8.3.635 Protein Sci 8:635-643 (1999)
PubMed id: 10091666  
 
 
Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris.
E.H.Rydberg, G.Sidhu, H.C.Vo, J.Hewitt, H.C.Côte, Y.Wang, S.Numao, R.T.MacGillivray, C.M.Overall, G.D.Brayer, S.G.Withers.
 
  ABSTRACT  
 
Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
18214874 C.A.Tarling, K.Woods, R.Zhang, H.C.Brastianos, G.D.Brayer, R.J.Andersen, and S.G.Withers (2008).
The search for novel human pancreatic alpha-amylase inhibitors: high-throughput screening of terrestrial and marine natural product extracts.
  Chembiochem, 9, 433-438.  
18552192 J.Y.Damián-Almazo, A.Moreno, A.López-Munguía, X.Soberón, F.González-Muñoz, and G.Saab-Rincón (2008).
Enhancement of the alcoholytic activity of alpha-amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by site-directed mutagenesis.
  Appl Environ Microbiol, 74, 5168-5177.  
17592362 R.Quezada-Calvillo, C.C.Robayo-Torres, Z.Ao, B.R.Hamaker, A.Quaroni, G.D.Brayer, E.E.Sterchi, S.S.Baker, and B.L.Nichols (2007).
Luminal substrate "brake" on mucosal maltase-glucoamylase activity regulates total rate of starch digestion to glucose.
  J Pediatr Gastroenterol Nutr, 45, 32-43.  
15722449 R.Maurus, A.Begum, H.H.Kuo, A.Racaza, S.Numao, C.Andersen, J.W.Tams, J.Vind, C.M.Overall, S.G.Withers, and G.D.Brayer (2005).
Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase.
  Protein Sci, 14, 743-755.
PDB codes: 1xgz 1xh0 1xh1 1xh2
15182367 N.Ramasubbu, C.Ragunath, P.J.Mishra, L.M.Thomas, G.Gyémánt, and L.Kandra (2004).
Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.
  Eur J Biochem, 271, 2517-2529.
PDB codes: 1jxj 1nm9
12834298 H.Akeboshi, Y.Kashiwagi, H.Aoki, T.Tonozuka, A.Nishikawa, and Y.Sakano (2003).
Construction of an efficient expression system for Aspergillus isopullulanase in Pichia pastoris, and a simple purification method.
  Biosci Biotechnol Biochem, 67, 1149-1153.  
10924103 A.M.Brzozowski, D.M.Lawson, J.P.Turkenburg, H.Bisgaard-Frantzen, A.Svendsen, T.V.Borchert, Z.Dauter, K.S.Wilson, and G.J.Davies (2000).
Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.
  Biochemistry, 39, 9099-9107.
PDB codes: 1e3x 1e3z 1e40 1e43
10769135 G.D.Brayer, G.Sidhu, R.Maurus, E.H.Rydberg, C.Braun, Y.Wang, N.T.Nguyen, C.M.Overall, and S.G.Withers (2000).
Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques.
  Biochemistry, 39, 4778-4791.
PDB codes: 1cpu 2cpu 3cpu
10640598 J.L.Cereghino, and J.M.Cregg (2000).
Heterologous protein expression in the methylotrophic yeast Pichia pastoris.
  FEMS Microbiol Rev, 24, 45-66.  
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.