PDBsum entry 1jxk

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protein metals links
Hydrolase PDB id
Protein chain
491 a.a. *
Waters ×323
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Role of ethe mobile loop in the mehanism of human salivary amylase
Structure: Alpha-amylase, salivary. Chain: a. Fragment: lacking the loop residues 306-310. Synonym: salivary alpha-amyalse. 1,4-alpha-d-glucan glucanohydrolase. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Organ: salivary glands. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108
1.90Å     R-factor:   0.176     R-free:   0.200
Authors: N.Ramasubbu,C.Ragunath,Z.Wang
Key ref:
N.Ramasubbu et al. (2003). Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase. J Mol Biol, 325, 1061-1076. PubMed id: 12527308 DOI: 10.1016/S0022-2836(02)01326-8
07-Sep-01     Release date:   14-Sep-01    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P04745  (AMY1_HUMAN) -  Alpha-amylase 1
511 a.a.
491 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.  - 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   3 terms 
  Biochemical function     catalytic activity     7 terms  


DOI no: 10.1016/S0022-2836(02)01326-8 J Mol Biol 325:1061-1076 (2003)
PubMed id: 12527308  
Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase.
N.Ramasubbu, C.Ragunath, P.J.Mishra.
Mammalian amylases harbor a flexible, glycine-rich loop 304GHGAGGA(310), which becomes ordered upon oligosaccharide binding and moves in toward the substrate. In order to probe the role of this loop in catalysis, a deletion mutant lacking residues 306-310 (Delta306) was generated. Kinetic studies showed that Delta306 exhibited: (1) a reduction (>200-fold) in the specific activity using starch as a substrate; (2) a reduction in k(cat) for maltopentaose and maltoheptaose as substrates; and (3) a twofold increase in K(m) (maltopentaose as substrate) compared to the wild-type (rHSAmy). More cleavage sites were observed for the mutant than for rHSAmy, suggesting that the mutant exhibits additional productive binding modes. Further insight into its role is obtained from the crystal structures of the two enzymes soaked with acarbose, a transition-state analog. Both enzymes modify acarbose upon binding through hydrolysis, condensation or transglycosylation reactions. Electron density corresponding to six and seven fully occupied subsites in the active site of rHSAmy and Delta306, respectively, were observed. Comparison of the crystal structures showed that: (1) the hydrophobic cover provided by the mobile loop for the subsites at the reducing end of the rHSAmy complex is notably absent in the mutant; (2) minimal changes in the protein-ligand interactions around subsites S1 and S1', where the cleavage would occur; (3) a well-positioned water molecule in the mutant provides a hydrogen bond interaction similar to that provided by the His305 in rHSAmy complex; (4) the active site-bound oligosaccharides exhibit minimal conformational differences between the two enzymes. Collectively, while the kinetic data suggest that the mobile loop may be involved in assisting the catalysis during the transition state, crystallographic data suggest that the loop may play a role in the release of the product(s) from the active site.
  Selected figure(s)  
Figure 6.
Figure 6. The overall polypeptide chain fold of the mutant D306:acarbose complex showing the various secondary binding sites of the ligand. Site 2 is anchored around residue Trp284 and is common between the rHSAmy and the D306 structures. This site (site 2) is about 5-6 Å away from the S3' subsite, suggesting a possibility that starch may bind with extended interactions along the surface of the molecule. Site 3 is anchored around Trp383 and is located near the A/C domain interface. An additional maltose residue (site 4) can be seen positioned suitably from the non-reducing end of the active site-bound moiety (site 1). Collectively, these sites give a preliminary view of the mode of starch binding with amylases.
Figure 7.
Figure 7. An illustration of the interactions formed by the ligands bound on the surface of amylase. This Figure was generated using HBPLUS[52.] and LIGPLOT. [53.] (a) Site 2 in the rHSAmy:acarbose complex; only three units were observable in the density maps corresponding to Agl and Glc. The Hmc unit is not seen in the density maps presumably because of disordering. (b) Site 2 in the D306:acarbose complex. Comparison of site 2 of the rHSAmy:acarbose and D306:acarbose complexes shows similarity in the binding site with residues Trp284, Tyr276 providing the stacking interactions. (c) Site 3 in the D306:acarbose complex. Trp383 provides the stacking interaction in site 3 in the mutant structure. In the structure of the D306:acarbose complex, unhydrolyzed acarbose units occupy sites 2 and 3.
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 325, 1061-1076) copyright 2003.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21261814 S.Cuyvers, E.Dornez, M.N.Rezaei, A.Pollet, J.A.Delcour, and C.M.Courtin (2011).
Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases.
  FEBS J, 278, 1098-1111.  
21397496 S.Park, S.Hyun, and J.Yu (2011).
Selective α-glucosidase substrates and inhibitors containing short aromatic peptidyl moieties.
  Bioorg Med Chem Lett, 21, 2441-2444.  
19422059 A.Pollet, E.Vandermarliere, J.Lammertyn, S.V.Strelkov, J.A.Delcour, and C.M.Courtin (2009).
Crystallographic and activity-based evidence for thumb flexibility and its relevance in glycoside hydrolase family 11 xylanases.
  Proteins, 77, 395-403.
PDB code: 3exu
18951906 C.Ragunath, S.G.Manuel, V.Venkataraman, H.B.Sait, C.Kasinathan, and N.Ramasubbu (2008).
Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-amylase in substrate hydrolysis and bacterial binding.
  J Mol Biol, 384, 1232-1248.  
19133500 J.E.Kerrigan, C.Ragunath, L.Kandra, G.Gyémánt, A.Lipták, L.Jánossy, J.B.Kaplan, and N.Ramasubbu (2008).
Modeling and biochemical analysis of the activity of antibiofilm agent Dispersin B.
  Acta Biol Hung, 59, 439-451.  
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.  
17044042 C.Albenne, L.K.Skov, V.Tran, M.Gajhede, P.Monsan, M.Remaud-Siméon, and G.André-Leroux (2007).
Towards the molecular understanding of glycogen elongation by amylosucrase.
  Proteins, 66, 118-126.  
17949435 S.G.Manuel, C.Ragunath, H.B.Sait, E.A.Izano, J.B.Kaplan, and N.Ramasubbu (2007).
Role of active-site residues of dispersin B, a biofilm-releasing beta-hexosaminidase from a periodontal pathogen, in substrate hydrolysis.
  FEBS J, 274, 5987-5999.  
17028947 F.Maczkowiak, and J.L.Da Lage (2006).
Origin and evolution of the Amyrel gene in the alpha-amylase multigene family of Diptera.
  Genetica, 128, 145-158.  
16907829 K.D.Saltzmann, K.A.Saltzmann, J.J.Neal, M.E.Scharf, and G.W.Bennett (2006).
Characterization of BGTG-1, a tergal gland-secreted alpha-amylase, from the German cockroach, Blattella germanica (L.).
  Insect Mol Biol, 15, 425-433.  
  16511271 S.Z.Fisher, L.Govindasamy, C.Tu, M.Agbandje-McKenna, D.N.Silverman, H.J.Rajaniemi, and R.McKenna (2006).
Structure of human salivary alpha-amylase crystallized in a C-centered monoclinic space group.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 62, 88-93.  
16294315 C.Hirtz, F.Chevalier, D.Centeno, V.Rofidal, J.C.Egea, M.Rossignol, N.Sommerer, and D.Deville de Périère (2005).
MS characterization of multiple forms of alpha-amylase in human saliva.
  Proteomics, 5, 4597-4607.  
16030022 X.Robert, R.Haser, H.Mori, B.Svensson, and N.Aghajari (2005).
Oligosaccharide binding to barley alpha-amylase 1.
  J Biol Chem, 280, 32968-32978.
PDB codes: 1rp8 1rp9 1rpk
15138257 A.Ohtaki, M.Mizuno, T.Tonozuka, Y.Sakano, and S.Kamitori (2004).
Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 2 with acarbose and cyclodextrins demonstrate the multiple substrate recognition mechanism.
  J Biol Chem, 279, 31033-31040.
PDB codes: 1vfk 1vfm 1vfo 1vfu 3a6o
15356864 G.André, and V.Tran (2004).
Putative implication of alpha-amylase loop 7 in the mechanism of substrate binding and reaction products release.
  Biopolymers, 75, 95.  
14573597 G.Golan, D.Shallom, A.Teplitsky, G.Zaide, S.Shulami, T.Baasov, V.Stojanoff, A.Thompson, Y.Shoham, and G.Shoham (2004).
Crystal structures of Geobacillus stearothermophilus alpha-glucuronidase complexed with its substrate and products: mechanistic implications.
  J Biol Chem, 279, 3014-3024.
PDB codes: 1k9d 1k9e 1k9f 1l8n 1mqp 1mqq 1mqr
15388967 K.Funane, T.Ishii, K.Terasawa, T.Yamamoto, and M.Kobayashi (2004).
Construction of chimeric glucansucrases for analyzing substrate-binding regions that affect the structure of glucan products.
  Biosci Biotechnol Biochem, 68, 1912-1920.  
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
14725761 T.Walma, J.Aelen, S.B.Nabuurs, M.Oostendorp, L.van den Berk, W.Hendriks, and G.W.Vuister (2004).
A closed binding pocket and global destabilization modify the binding properties of an alternatively spliced form of the second PDZ domain of PTP-BL.
  Structure, 12, 11-20.
PDB code: 1ozi
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 code is shown on the right.