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PDBsum entry 1lnb
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Hydrolase (metalloprotease) PDB id
1lnb

 

 

 

 

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Contents
Protein chain
316 a.a. *
Ligands
VAL-LYS
DMS
Metals
_FE
_CA ×4
Waters ×159
* Residue conservation analysis
PDB id:
1lnb
Name: Hydrolase (metalloprotease)
Title: A structural analysis of metal substitutions in thermolysin
Structure: Thermolysin. Chain: e. Engineered: yes
Source: Bacillus thermoproteolyticus. Organism_taxid: 1427
Biol. unit: Dimer (from PQS)
Resolution:
1.80Å     R-factor:   0.152    
Authors: D.R.Holland,A.C.Hausrath,D.Juers,B.W.Matthews
Key ref: D.R.Holland et al. (1995). Structural analysis of zinc substitutions in the active site of thermolysin. Protein Sci, 4, 1955-1965. PubMed id: 8535232 DOI: 10.1002/pro.5560041001
Date:
13-May-94     Release date:   08-May-95    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P00800  (THER_BACTH) -  Thermolysin from Bacillus thermoproteolyticus
Seq:
Struc: 		 
Seq:
Struc: 		548 a.a.
316 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.3.4.24.27  - thermolysin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Preferential cleavage: Xaa-|-Leu > Xaa-|-Phe.
      Cofactor: Ca(2+); Zn(2+)

 

 
DOI no: 10.1002/pro.5560041001 Protein Sci 4:1955-1965 (1995)
PubMed id: 8535232  
 
 
Structural analysis of zinc substitutions in the active site of thermolysin.
D.R.Holland, A.C.Hausrath, D.Juers, B.W.Matthews.
 
  ABSTRACT  
 
Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
22477786 A.Thorn, and G.M.Sheldrick (2011).
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20459833 D.H.Bryant, M.Moll, B.Y.Chen, V.Y.Fofanov, and L.E.Kavraki (2010).
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Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease.
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19359249 M.Y.Zakharova, N.A.Kuznetsov, S.A.Dubiley, A.V.Kozyr, O.S.Fedorova, D.M.Chudakov, D.G.Knorre, I.G.Shemyakin, A.G.Gabibov, and A.V.Kolesnikov (2009).
Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches.
  J Biol Chem, 284, 17902-17913.  
19152630 O.A.Adekoya, and I.Sylte (2009).
The thermolysin family (m4) of enzymes: therapeutic and biotechnological potential.
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20161624 R.Wu, P.Hu, S.Wang, Z.Cao, and Y.Zhang (2009).
Flexibility of Catalytic Zinc Coordination in Thermolysin and HDAC8: A Born-Oppenheimer ab initio QM/MM Molecular Dynamics Study.
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19332551 S.Wydau, G.van der Rest, C.Aubard, P.Plateau, and S.Blanquet (2009).
Widespread Distribution of Cell Defense against D-Aminoacyl-tRNAs.
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18041759 B.Seebeck, I.Reulecke, A.Kämper, and M.Rarey (2008).
Modeling of metal interaction geometries for protein-ligand docking.
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  18959747 L.A.Bruce, J.A.Sigman, D.Randall, S.Rodriguez, M.M.Song, Y.Dai, D.E.Elmore, A.Pabon, M.J.Glucksman, and A.J.Wolfson (2008).
Hydrogen bond residue positioning in the 599-611 loop of thimet oligopeptidase is required for substrate selection.
  FEBS J, 275, 5607-5617.  
16790782 C.Kooi, B.Subsin, R.Chen, B.Pohorelic, and P.A.Sokol (2006).
Burkholderia cenocepacia ZmpB is a broad-specificity zinc metalloprotease involved in virulence.
  Infect Immun, 74, 4083-4093.  
17096442 I.Bertini, V.Calderone, M.Fragai, C.Luchinat, M.Maletta, and K.J.Yeo (2006).
Snapshots of the reaction mechanism of matrix metalloproteinases.
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PDB codes: 2oxu 2oxw 2oxz 2oy2 2oy4
16432573 S.Siemann, H.R.Badiei, V.Karanassios, T.Viswanatha, and G.I.Dmitrienko (2006).
68Zn isotope exchange experiments reveal an unusual kinetic lability of the metal ions in the di-zinc form of IMP-1 metallo-beta-lactamase.
  Chem Commun (Camb), (), 532-534.  
16199560 A.K.Chang, H.Y.Kim, J.E.Park, P.Acharya, I.S.Park, S.M.Yoon, H.J.You, K.S.Hahm, J.K.Park, and J.S.Lee (2005).
Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis.
  J Bacteriol, 187, 6909-6916.  
16215833 K.Yoshimune, A.Hirayama, and M.Moriguchi (2005).
A metal ion as a cofactor attenuates substrate inhibition in the enzymatic production of a high concentration of D-glutamate using N-acyl-D-glutamate amidohydrolase.
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16137162 M.Albrecht, and P.Stortz (2005).
Metallacyclopeptides: artificial analogues of naturally occurring peptides.
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16322579 T.A.Binkowski, A.Joachimiak, and J.Liang (2005).
Protein surface analysis for function annotation in high-throughput structural genomics pipeline.
  Protein Sci, 14, 2972-2981.  
15508121 A.S.Galanis, G.A.Spyroulias, G.Pairas, E.Manessi-Zoupa, and P.Cordopatis (2004).
Solid-phase synthesis and conformational properties of angiotensin converting enzyme catalytic-site peptides: the basis for a structural study on the enzyme-substrate interaction.
  Biopolymers, 76, 512-526.  
12767125 A.S.Galanis, G.A.Spyroulias, R.Pierattelli, A.Tzakos, A.Troganis, I.P.Gerothanassis, G.Pairas, E.Manessi-Zoupa, and P.Cordopatis (2003).
Zinc binding in peptide models of angiotensin-I converting enzyme active sites studied through 1H-NMR and chemical shift perturbation mapping.
  Biopolymers, 69, 244-252.  
12833153 B.E.Coggins, X.Li, A.L.McClerren, O.Hindsgaul, C.R.Raetz, and P.Zhou (2003).
Structure of the LpxC deacetylase with a bound substrate-analog inhibitor.
  Nat Struct Biol, 10, 645-651.
PDB code: 1nzt
12819349 D.A.Whittington, K.M.Rusche, H.Shin, C.A.Fierke, and D.W.Christianson (2003).
Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis.
  Proc Natl Acad Sci U S A, 100, 8146-8150.
PDB code: 1p42
12711857 H.L.Liu, Y.Ho, and C.M.Hsu (2003).
The effect of metal ions on the binding of ethanol to human alcohol dehydrogenase beta2beta2.
  J Biomed Sci, 10, 302-312.  
12037302 A.C.Hausrath, and B.W.Matthews (2002).
Thermolysin in the absence of substrate has an open conformation.
  Acta Crystallogr D Biol Crystallogr, 58, 1002-1007.
PDB code: 1l3f
12070326 C.Hetényi, and D.van der Spoel (2002).
Efficient docking of peptides to proteins without prior knowledge of the binding site.
  Protein Sci, 11, 1729-1737.  
12044150 L.Meng, S.Ruebush, V.M.D'souza, A.J.Copik, S.Tsunasawa, and R.C.Holz (2002).
Overexpression and divalent metal binding properties of the methionyl aminopeptidase from Pyrococcus furiosus.
  Biochemistry, 41, 7199-7208.  
12364591 N.Shomron, H.Malca, I.Vig, and G.Ast (2002).
Reversible inhibition of the second step of splicing suggests a possible role of zinc in the second step of splicing.
  Nucleic Acids Res, 30, 4127-4137.  
11274446 G.Loussouarn, L.R.Phillips, R.Masia, T.Rose, and C.G.Nichols (2001).
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  Proc Natl Acad Sci U S A, 98, 4227-4232.  
11241598 H.Strasdeit (2001).
The First Cadmium-Specific Enzyme.
  Angew Chem Int Ed Engl, 40, 707-709.  
11148046 J.E.Jackman, C.R.Raetz, and C.A.Fierke (2001).
Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site.
  Biochemistry, 40, 514-523.  
11559826 M.Pallaoro, A.Lahm, G.Biasiol, M.Brunetti, C.Nardella, L.Orsatti, F.Bonelli, S.Orrù, F.Narjes, and C.Steinkühler (2001).
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  J Virol, 75, 9939-9946.  
11553770 M.T.Hilgers, and M.L.Ludwig (2001).
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  Proc Natl Acad Sci U S A, 98, 11169-11174.
PDB code: 1ie0
10735252 A.Coffey, B.van den Burg, R.Veltman, and T.Abee (2000).
Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes.
  J Appl Microbiol, 88, 132-141.  
10924116 B.C.Tripp, and J.G.Ferry (2000).
A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila.
  Biochemistry, 39, 9232-9240.  
10924115 T.M.Iverson, B.E.Alber, C.Kisker, J.G.Ferry, and D.C.Rees (2000).
A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila.
  Biochemistry, 39, 9222-9231.
PDB codes: 1qq0 1qre 1qrf 1qrg 1qrl 1qrm
10736182 V.M.D'souza, B.Bennett, A.J.Copik, and R.C.Holz (2000).
Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli.
  Biochemistry, 39, 3817-3826.  
10651278 A.C.English, S.H.Done, L.S.Caves, C.R.Groom, and R.E.Hubbard (1999).
Locating interaction sites on proteins: the crystal structure of thermolysin soaked in 2% to 100% isopropanol.
  Proteins, 37, 628-640.
PDB codes: 1tli 1tlx 2tli 2tlx 3tli 4tli 5tli 6tli 7tli 8tli
10074338 B.diSioudi, J.K.Grimsley, K.Lai, and J.R.Wild (1999).
Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity.
  Biochemistry, 38, 2866-2872.  
  10595562 K.S.Makarova, and N.V.Grishin (1999).
Thermolysin and mitochondrial processing peptidase: how far structure-functional convergence goes.
  Protein Sci, 8, 2537-2540.  
10328266 S.M.King, and W.C.Johnson (1999).
Assigning secondary structure from protein coordinate data.
  Proteins, 35, 313-320.  
10545376 U.Ryde (1999).
Carboxylate binding modes in zinc proteins: A theoretical study
  Biophys J, 77, 2777-2787.  
9665723 E.G.Orellano, J.E.Girardini, J.A.Cricco, E.A.Ceccarelli, and A.J.Vila (1998).
Spectroscopic characterization of a binuclear metal site in Bacillus cereus beta-lactamase II.
  Biochemistry, 37, 10173-10180.  
9928125 E.Pauthe, M.Dauchez, H.Berry, M.Berjot, J.P.Monti, A.J.Alix, and V.Larreta-Garde (1998).
Enzymatic and structural approaches of the thermolysin mechanism in glycerol-containing media.
  Ann N Y Acad Sci, 864, 458-462.  
9548762 O.R.Veltman, V.G.Eijsink, G.Vriend, A.de Kreij, G.Venema, and B.Van den Burg (1998).
Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.
  Biochemistry, 37, 5305-5311.  
  9041649 O.Bogin, M.Peretz, and Y.Burstein (1997).
Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids.
  Protein Sci, 6, 450-458.  
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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