Structural insights into substrate specificity of crotonase from the n-butanol producing bacterium Clostridium acetobutylicum.

Abstract

Crotonase from Clostridium acetobutylicum (CaCRT) is an enzyme that catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA in the n-butanol biosynthetic pathway. To investigate the molecular mechanism underlying n-butanol biosynthesis, we determined the crystal structures of the CaCRT protein in apo- and acetoacetyl-CoA bound forms. Similar to other canonical crotonase enzymes, CaCRT forms a hexamer by the dimerization of two trimers. A crystal structure of CaCRT in complex with acetoacetyl-CoA revealed that Ser69 and Ala24 to be signature residues of CaCRT, which results in a distinct ADP binding mode wherein the ADP moiety is bound at a different position compared with other crotonases. We also revealed that the substrate specificity of crotonase enzymes is determined by both the structural feature of the α3 helix region and the residues contributing the enoyl-CoA binding pocket. A tight formed α3 helix and two phenylalanine residues, Phe143 and Phe233, aid CaCRT to accommodate crotonyl-CoA as the substrate. The key residues involved in substrate binding, enzyme catalysis and substrate specificity were confirmed by site-directed mutagenesis.

• Authors: Eun-Jung Kim, Yeo-Jin Kim, Kyung-Jin Kim
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 451 Issue 3 Pg. 431-5 (Aug 29 2014) ISSN: 1090-2104 [Electronic] United States
• PMID: 25110148 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical References

• Acyl Coenzyme A
• acetoacetyl CoA
• crotonyl-coenzyme A
• Enoyl-CoA Hydratase

Topics

• Acyl Coenzyme A (metabolism)
• Amino Acid Sequence
• Catalysis
• Catalytic Domain
• Clostridium acetobutylicum (enzymology)
• Crystallography, X-Ray
• Enoyl-CoA Hydratase (chemistry, metabolism)
• Models, Molecular
• Molecular Sequence Data
• Protein Multimerization
• Protein Structure, Secondary
• Substrate Specificity