Abstract

Multifunctional enzyme, type‐1 (MFE1) 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 MFE1 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 MFE1 are correlated. The possible importance of the conformational flexibility of MFE1 for its biocatalytic properties is discussed.