PDBsum entry 2tmn

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Hydrolase/hydrolase inhibitor PDB id
Protein chain
316 a.a. *
_CA ×4
Waters ×168
* Residue conservation analysis
PDB id:
Name: Hydrolase/hydrolase inhibitor
Title: Crystallographic structural analysis of phosphoramidates as and transition-state analogs of thermolysin
Structure: Thermolysin. Chain: e. Ec:
Source: Bacillus thermoproteolyticus. Organism_taxid: 1427
Biol. unit: Tetramer (from PQS)
1.60Å     R-factor:   0.179    
Authors: D.E.Tronrud,A.F.Monzingo,B.W.Matthews
Key ref: D.E.Tronrud et al. (1986). Crystallographic structural analysis of phosphoramidates as inhibitors and transition-state analogs of thermolysin. Eur J Biochem, 157, 261-268. PubMed id: 3709536
29-Jun-87     Release date:   09-Jan-89    
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Protein chain
Pfam   ArchSchema ?
P00800  (THER_BACTH) -  Thermolysin
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.  - Thermolysin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Preferential cleavage: Xaa-|-Leu > Xaa-|-Phe.
      Cofactor: Ca(2+); Zn(2+)
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     proteolysis   1 term 
  Biochemical function     metalloendopeptidase activity     1 term  


Eur J Biochem 157:261-268 (1986)
PubMed id: 3709536  
Crystallographic structural analysis of phosphoramidates as inhibitors and transition-state analogs of thermolysin.
D.E.Tronrud, A.F.Monzingo, B.W.Matthews.
The mode of binding to thermolysin of the unsubstituted phosphoramidate inhibitor N-phosphoryl-L-leucinamide (P-Leu-NH2) has been determined crystallographically and refined at high resolution (R = 17.9% to 0.16-nm resolution). The mode of binding of the naturally occurring thermolysin inhibitor phosphoramidon reported previously [Weaver, L. H., Kester, W. R. and Matthews, B. W. (1977) J. Mol. Biol. 114, 119-132] has also been confirmed by crystallographic refinement (R = 17.4% to 0.23-nm resolution). Phosphoramidon binds to the enzyme with a single oxygen of the phosphoramidate moiety as a zinc ligand. Together with three ligands to the metal from the protein the resultant complex has approximately tetrahedral geometry. However, in the case of P-Leu-NH2, two of the phosphoramidate oxygens interact with the zinc to form a complex that tends towards pentacoordinate. In this respect, P-Leu-NH2 appears to be a better transition-state analog than is phosphoramidon. In addition, the phosphorus-nitrogen bond length in P-Leu-NH2 is 0.18 nm, suggesting that the nitrogen is protonated whereas the same bond in phosphoramidon is much shorter (0.15 nm) suggesting that the nitrogen does not carry a charge. In phosphoramidon the distance from the phosphoramide nitrogen to Glu-143 is 0.39 nm whereas in P-Leu-NH2 this distance decreases to 0.34 nm. Taken together, these observations provide additional evidence in support of the participation of pentacoordinate intermediates in the mechanism of action of thermolysin [Holmes, M. A. and Matthews, B. W. (1981) Biochemistry 20, 6912-6920] and the role of Glu-143 in first promoting the attack of a water molecule on the carbonyl carbon of the scissile bond and subsequently acting as a 'proton shuttle' to transfer the proton to the leaving nitrogen [Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5724-5729; Hangauer, D. G., Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5730-5741].

Literature references that cite this PDB file's key reference

  PubMed id Reference
23275160 A.Ruf, M.Stihle, J.Benz, M.Schmidt, and H.Sobek (2013).
Structure of Gentlyase, the neutral metalloprotease of Paenibacillus polymyxa.
  Acta Crystallogr D Biol Crystallogr, 69, 24-31.
PDB codes: 4b52 4ger
19181663 B.B.Xie, F.Bian, X.L.Chen, H.L.He, J.Guo, X.Gao, Y.X.Zeng, B.Chen, B.C.Zhou, and Y.Z.Zhang (2009).
Cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics: new insights into relationship between conformational flexibility and hydrogen bonding.
  J Biol Chem, 284, 9257-9269.  
18974160 M.Kusano, K.Yasukawa, and K.Inouye (2009).
Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability.
  J Biochem, 145, 103-113.  
19152630 O.A.Adekoya, and I.Sylte (2009).
The thermolysin family (m4) of enzymes: therapeutic and biotechnological potential.
  Chem Biol Drug Des, 73, 7.  
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.
  J Chem Theory Comput, 6, 337.  
17516427 E.Proschak, M.Rupp, S.Derksen, and G.Schneider (2008).
Shapelets: possibilities and limitations of shape-based virtual screening.
  J Comput Chem, 29, 108-114.  
17429823 B.M.McArdle, and R.J.Quinn (2007).
Identification of protein fold topology shared between different folds inhibited by natural products.
  Chembiochem, 8, 788-798.  
16465317 J.Wang, M.Uttamchandani, L.P.Sun, and S.Q.Yao (2006).
Activity-based high-throughput profiling of metalloprotease inhibitors using small molecule microarrays.
  Chem Commun (Camb), (), 717-719.  
15526325 M.Kontoyianni, G.S.Sokol, and L.M.McClellan (2005).
Evaluation of library ranking efficacy in virtual screening.
  J Comput Chem, 26, 11-22.  
15027050 S.Swaminathan, S.Eswaramoorthy, and D.Kumaran (2004).
Structure and enzymatic activity of botulinum neurotoxins.
  Mov Disord, 19, S17-S22.  
12832763 M.Selkti, A.Tomas, J.F.Gaucher, T.Prangé, M.C.Fournié-Zaluski, H.Chen, and B.P.Roques (2003).
Interactions of a new alpha-aminophosphinic derivative inside the active site of TLN (thermolysin): a model for zinc-metalloendopeptidase inhibition.
  Acta Crystallogr D Biol Crystallogr, 59, 1200-1205.
PDB codes: 1no0 1os0
11559368 Kreij, B.van den Burg, O.R.Veltman, G.Vriend, G.Venema, and V.G.Eijsink (2001).
The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.
  Eur J Biochem, 268, 4985-4991.  
10930836 G.E.Dale, B.D'Arcy, C.Yuvaniyama, B.Wipf, C.Oefner, and A.D'Arcy (2000).
Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastoris.
  Acta Crystallogr D Biol Crystallogr, 56, 894-897.  
10694409 L.Li, T.Binz, H.Niemann, and B.R.Singh (2000).
Probing the mechanistic role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain.
  Biochemistry, 39, 2399-2405.  
8652513 W.L.Mock, and D.J.Stanford (1996).
Arazoformyl dipeptide substrates for thermolysin. Confirmation of a reverse protonation catalytic mechanism.
  Biochemistry, 35, 7369-7377.  
7622493 A.Beaumont, M.J.O'Donohue, N.Paredes, N.Rousselet, M.Assicot, C.Bohuon, M.C.Fournié-Zaluski, and B.P.Roques (1995).
The role of histidine 231 in thermolysin-like enzymes. A site-directed mutagenesis study.
  J Biol Chem, 270, 16803-16808.  
7561976 C.McMartin, and R.S.Bohacek (1995).
Flexible matching of test ligands to a 3D pharmacophore using a molecular superposition force field: comparison of predicted and experimental conformations of inhibitors of three enzymes.
  J Comput Aided Mol Des, 9, 237-250.  
7738608 G.Klebe, T.Mietzner, and F.Weber (1994).
Different approaches toward an automatic structural alignment of drug molecules: applications to sterol mimics, thrombin and thermolysin inhibitors.
  J Comput Aided Mol Des, 8, 751-778.  
7964924 R.L.DesJarlais, and J.S.Dixon (1994).
A shape- and chemistry-based docking method and its use in the design of HIV-1 protease inhibitors.
  J Comput Aided Mol Des, 8, 231-242.  
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.  
8081834 M.Izquierdo-Martin, and R.L.Stein (1993).
Mechanistic studies on the inhibition of stromelysin by a peptide phosphonamidate.
  Bioorg Med Chem, 1, 19-26.  
1730223 S.Mangani, P.Carloni, and P.Orioli (1992).
X-ray diffraction study of the interaction between carboxypeptidase A and (S)-(+)-1-amino-2-phenylethyl phosphonic acid.
  Eur J Biochem, 203, 173-177.  
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.