Crystal structure of liganded rat peroxisomal multifunctional enzyme type 1: a flexible molecule with two interconnected active sites

• Published in: The Journal of biological chemistry 2010-07, Vol.285 (31), p.24089-24098
• Main Authors: Kasaragod, Prasad, Venkatesan, Rajaram, Kiema, Tiila R, Hiltunen, J Kalervo, Wierenga, Rik K
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Catalytic Domain; Crystallography, X-Ray - methods; Enzymology; Escherichia coli - enzymology; Fatty Acids - chemistry; Kinetics; Liver - enzymology; Molecular Sequence Data; Multienzyme Complexes - chemistry; Oxygen - chemistry; Peroxisomes - metabolism; Protein Conformation; Protein Folding; Protein Structure and Folding; Rats
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.117606

The crystal structure of the full-length rat peroxisomal multifunctional enzyme, type 1 (rpMFE1), has been determined at 2.8 A resolution. This enzyme has three catalytic activities and two active sites. The N-terminal part has the crotonase fold, which builds the active site for the Delta(3),Delta(2)-enoyl-CoA isomerase and the Delta(2)-enoyl-CoA hydratase-1 catalytic activities, and the C-terminal part has the (3S)-hydroxyacyl-CoA dehydrogenase fold and makes the (3S)-hydroxyacyl-CoA dehydrogenase active site. rpMFE1 is a multidomain protein having five domains (A-E). The crystal structure of full-length rpMFE1 shows a flexible arrangement of the A-domain with respect to the B-E-domains. Because of a hinge region near the end of the A-domain, two different positions of the A-domain were observed for the two protein molecules (A and B) of the asymmetric unit. In the most closed conformation, the mode of binding of CoA is stabilized by domains A and B (helix-10), as seen in other crotonase fold members. Domain B, although functionally belonging to the N-terminal part, is found tightly associated with the C-terminal part, i.e. fixed to the E-domain. The two active sites of rpMFE1 are approximately 40 A apart, separated by a tunnel, characterized by an excess of positively charged side chains. Comparison of the structures of rpMFE1 with the monofunctional crotonase and (3S)-hydroxyacyl-CoA dehydrogenase superfamily enzymes, as well as with the bacterial alpha(2)beta(2)-fatty acid oxidation multienzyme complex, reveals that this tunnel could be important for substrate channeling, as observed earlier on the basis of the kinetics of rpMFE1 purified from rat liver.