1U4N

Crystal Structure Analysis of the M211S/R215L EST2 mutant

Summary for 1U4N

• Entry DOI: 10.2210/pdb1u4n/pdb
• Related: 1EVQ 1QZ3
• Descriptor: CARBOXYLESTERASE EST2, SULFATE ION (3 entities in total)
• Functional Keywords: alpha/beta hydrolase fold, hydrolase
• Biological source: Alicyclobacillus acidocaldarius
• Total number of polymer chains: 1
• Total formula weight: 34346.88

Authors

De Simone, G.,Menchise, V.,Alterio, V.,Mandrich, L.,Rossi, M.,Manco, G.,Pedone, C. (deposition date: 2004-07-26, release date: 2004-10-05, Last modification date: 2023-08-23)

Primary citation

De Simone, G.,Menchise, V.,Alterio, V.,Mandrich, L.,Rossi, M.,Manco, G.,Pedone, C.
The Crystal Structure of an EST2 Mutant Unveils Structural Insights on the H Group of the Carboxylesterase/Lipase Family.
J.Mol.Biol., 343:137-146, 2004

PubMed Abstract: Esterase 2 (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius is a thermostable serine hydrolase belonging to the H group of the esterase/lipase family. This enzyme hydrolyzes monoacylesters of different acyl-chain length and various compounds with industrial interest. EST2 displays an optimal temperature at 70 degrees C and maximal activity with pNP-esters having acyl-chain bearing from six to eight carbon atoms. EST2 mutants with different substrate specificity were also designed, generated by site-directed mutagenesis, and biochemically characterized. To better define at structural level the enzyme reaction mechanism, a crystallographic analysis of one of these mutants, namely M211S/R215L, was undertaken. Here we report its three-dimensional structure at 2.10A resolution. Structural analysis of the enzyme revealed an unexpected dimer formation as a consequence of a domain-swapping event involving its N-terminal region. This phenomenon was absent in the case of the enzyme bound to an irreversible inhibitor having optimal substrate structural features. A detailed comparison of the enzyme structures before and following binding to this molecule showed a movement of the N-terminal helices resulting from a trans-cis isomerization of the F37-P38 peptide bond. These findings suggest that this carboxylesterase presents two distinct structural arrangements reminiscent of the open and closed forms already reported for lipases. Potential biological implications associated with the observed quaternary reorganization are here discussed in light of the biochemical properties of other lipolytic members of the H group.

PubMed: 15381425
DOI: 10.1016/j.jmb.2004.08.014

Experimental method

X-RAY DIFFRACTION (2.1 Å)