Hydratase Activities of Green Fluorescent Protein Tagged Human Multifunctional Enzyme Type 2 Hydratase Domain and its Variants

Abstract

INTRODUCTION Human peroxisomal multifunctional enzyme (MFE) type 1 (MFE1) , often called L-bifunctional protein (LBP) , and MFE2, often called 17-β-hydroxysteroid dehydrogenase type 4 (HSD17B4) or D-bifunctional protein (DBP) , catalyze the hydration of trans-2-enoyl-coenzyme A (CoA) and the subsequent dehydrogenation of 3-hydroxyacyl-CoA in peroxisomal fatty acid β-oxidation 1-3). Although both MFE1 and MFE2 possess hydratase and dehydrogenase activities, their molecular structures differ 3). It is known that MFE1 and MFE2 yield 3S-and 3R-hydroxyacyl-CoA from trans-2-enoyl-CoA, respectively (Fig. 1) ; however, the physiological significance of this distinction is not clear. Deficiency of MFE2 causes fatal lipid metabolism disorders 4-8) , whereas the consequences of MFE1 deficiency remain unclear. Thus, it is considered that MFE2 is very important for the maintenance of life. It is anticipated that more detail

Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity

“…A plasmid encoding GFP-MFE2H (pGFP-MFE2H) [7] was used as a template for site-directed mutagenesis [9]. The substrate ( trans -2-hexadecenoyl-CoA) and the product (3 R -hydroxyhexadecanoyl-CoA) were synthesized chemically as described previously [6].…”

“…After digestion of template DNA (pGFP-MFE2H) with 2 units of Dpn I (37 °C for 1 h), constructed plasmids were introduced into competent cells of Escherichia coli JM109. The transformation of E. coli JM109 and expression and purification of GFP-MFE2H variants were performed as described in our previous report [7].…”

“…Incubation and HPLC were performed as described in our previous report [7] as follows: 40 μL of 100 μM trans -2-hexadecenoyl-CoA was added to 10 μL of enzyme solution (15–25 units) and incubated at 30 °C for 10 min. To stop the reaction, the mixture was placed on ice and 10 μL of 0.1 M HCl was added.…”

“…This method enables us to analyze the activity of hydratase even if d -BP and l -BP are both present in the experimental system. In addition, we have cloned human d -BP hydratase domain and expressed it as a fusion protein with green fluorescent protein (GFP), which we named GFP-MFE2H, to demonstrate the utility of the HPLC method [7]. This fusion protein is easy to purify and assay because of its traceability under ultraviolet (UV) light.…”

“…Black and red indicate amino acids with conservation rates > 70%. Black indicates amino acids for which hydratase activities of variants have already been investigated [7]. Red indicates amino acids for which the effects of missense mutations are not clear.…”