Structural enzymology comparisons of multifunctional enzyme, type‐1 (MFE1): the flexibility of its dehydrogenase part

Abstract

Multifunctional enzyme, type‐1 ( MFE 1) is a monomeric enzyme with a 2E‐enoyl‐CoA hydratase and a 3S‐hydroxyacyl‐CoA dehydrogenase ( HAD ) active site. Enzyme kinetic data of rat peroxisomal MFE 1 show that the catalytic efficiencies for converting the short‐chain substrate 2E‐butenoyl‐CoA into acetoacetyl‐CoA are much lower when compared with those of the homologous monofunctional enzymes. The mode of binding of acetoacetyl‐CoA (to the hydratase active site) and the very similar mode of binding of NAD + and NADH (to the HAD part) are described and compared with those of their monofunctional counterparts. Structural comparisons suggest that the conformational flexibility of the HAD and hydratase parts of MFE 1 are correlated. The possible importance of the conformational flexibility of MFE 1 for its biocatalytic properties is discussed. Database Structural data are available in PDB database under the accession number 5MGB .

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

“…To compare the structures, the SSM protocol (Krissinel & Henrick, 2004) of Coot was used to superimpose the structures of the monofunctional homologues on the structures of RnMFE1, whereas the LSQ option of Coot (using only the C atoms) was used to compare the MFE1 structures with each other. For the latter comparison, the MFE1 structure was used which was obtained by cocrystallization in the presence of 2 mM AcAc-CoA and 2 mM NAD + , with AcAc-CoA bound in the ECH active site and NAD + bound in the HAD active site of both molecules in the asymmetric unit (PDB entry 5mgb, 2.8 Å resolution; Kasaragod et al, 2017). For comparison with the monofunctional homologues two structures were used: the structure of the rat mitochondrial 2E-enoyl-CoA hydratase (RnECH) complexed with AcAc-CoA (PDB entry 1dub, 2.5 Å resolution; Engel et al, 1996) and the structure of the human mitochondrial 3S-hydroxyacyl-CoA dehydrogenase (HsHAD) complexed with AcAc-CoA and NAD + (PDB entry 1f0y, 1.8 Å resolution; Barycki et al, 2000).…”

“…Previous structural studies on MFE1 have resulted in the crystal structure of the truncated BCDE construct (lacking the A domain; Taskinen et al, 2006), as well as the structures of various complexes of wild-type MFE1, providing insight into the mode of binding of 3S-hydroxyacyl-CoA and acetoacetyl-CoA (AcAc-CoA) to the ECH active site and of NAD + and NADH to the HAD active site. These studies have also highlighted the existence of two hinge motions (Kasaragod et al, 2017): between the A domain and the HAD part, and between the C domain and the D/E domains.…”

“…The best-studied monofunctional HAD enzyme is the human mitochondrial enzyme, HsHAD, which is a dimer. The sequence identity between the HAD part of RnMFE1 and HsHAD is 31% (Kasaragod et al, 2017). In the active site a proton and a hydride are abstracted from the 3S-hydroxyacyl-CoA substrate, and are transferred to the catalytic histidine (His431 in MFE1; Fig.…”

“…The thioester-pantetheine moiety is hydrogen-bonded to the C domain by hydrogen bonds from the thioester O atom to the N atom of Asn161 and of N4P to the O atom of Asn161 in the CB6 -strand. Other van der Waals interactions and water-mediated hydrogen bonds of the pantetheine moiety are made with residues of the -hairpin loop between CB6 and CB7 of the C domain and with residues of the DH2-DH3 loops of both dimerization domains, which together form the pantetheine-binding tunnel (Kasaragod et al, 2017). The HsHAD dimerization domain corresponds to the D domain of MFE1.…”

“…Comparison of the structures of these two molecules shows the conformational flexibility of MFE1, in particular of the A and C domains with respect to the D/E domains. From an analysis of the distances between carefully selected C atoms in the various domains, it has been found that in molecule A the A domain is in an open conformation with respect to the HAD part and the C domain is in a more closed conformation with respect to the D/E domain, whereas in molecule B the A domain is more closed and the C domain is more open (Kasaragod et al, 2017). The conformational flexibility properties have been further studied here using RnMFE1 crystallized in a different crystal form (that diffracts much better than the regular crystal form), as well as by carrying out further crystallographic binding studies with the regular crystal form.…”