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Crystallographic binding studies of rat peroxisomal multifunctional enzyme type 1 with 3-ketodecanoyl-CoA: capturing active and inactive states of its hydratase and dehydrogenase catalytic sites

DOI: 10.1107/S2059798320013819 DOI Help

Authors: Shruthi Sridhar (University of Oulu) , Werner Schmitz (University of Wurzburg) , J. Kalervo Hiltunen (University of Oulu) , Rajaram Venkatesan (University of Oulu) , Ulrich Bergmann (University of Oulu) , Tiila-Riikka Kiema (University of Oulu) , Rikkert K. Wierenga (University of Oulu)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Structural Biology , VOL 76 , PAGES 1256 - 1269

State: Published (Approved)
Published: December 2020
Diamond Proposal Number(s): 26302 , 24732

Abstract: The peroxisomal multifunctional enzyme type 1 (MFE1) catalyzes two successive reactions in the β-oxidation cycle: the 2E-enoyl-CoA hydratase (ECH) and NAD+-dependent 3S-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 Å resolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD+ bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part.

Journal Keywords: peroxisomal multifunctional enzyme type 1; Rossmann fold; reaction mechanism; closed active site; fatty-acid oxidation; conformational flexibility

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography

Other Facilities: ID23-EH1, ID14-4 at ESRF

Added On: 09/12/2020 09:10

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Organic Chemistry Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)