Abstract Mammalian peroxisomes contain two parallel multifunctional enzymes (MFE), MFE type 1 and MFE type 2 (MFE-2), which are responsible for the degradation of fatty acids. They both catalyze the second and third reactions of the β-oxidation pathway, but through reciprocal stereochemical courses. MFE-2 possesses (2E)-enoyl-CoA hydratase-2 and (3R)-hydroxyacyl-CoA dehydrogenase activities. In addition, the carboxy-terminal part is similar to the sterol carrier protein type 2 (SCP-2). The purpose of this work was to study the structure-function relationship of functional domains of mammalian MFE-2 by recombinant DNA technology, enzyme kinetics and X-ray crystallography. The work started with the identification of conserved regions in MFE-2. This information was utilized when dehydrogenase, hydratase-2 and/or SCP-2-like domain were produced as separate recombinant proteins. Subsequently, both dehydrogenase and SCP-2-like domains were crystallized and their crystal structures were solved. The structure of the dehydrogenase region of rat MFE-2 contains the basic α/β short-chain alcohol dehydrogenase/reductase (SDR) fold and the four-helix bundle at the dimer interface, which is typical of dimeric SDR enzymes. However, the structure has a novel carboxy-terminal domain not seen among the known structures. This domain lines the active site cavity of the neighbouring monomer, reflecting cooperative behaviour within a homodimer. The monomeric SCP-2-like domain of human MFE-2 has the same fold as rabbit SCP-2. The structure includes a hydrophobic tunnel occupied by an ordered Triton X-100 molecule, demonstrating the ligand-binding site. Compared to the unliganded rabbit SCP-2 structure, the position of the carboxy-terminal helix is different. The movement of this helix in the liganded human SCP-2-like domain resulted in the exposure of a peroxisomal targeting signal, suggesting ligand-assisted protein import into peroxisomes. The roles of conserved protic residues in the hydratase-2 region of human MFE-2 were studied by mutating them to alanine. In the first step, the ability of mutated variants to utilize oleic acid in vivo was tested with Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2). Subsequently, in vitro characterization of the mutant enzymes revealed two amino acid residues, Glu366 and Asp510, vital for hydratase-2 activity. The results indicate that the acid-base catalysis is valid for hydratase-2.

Expressing foreign proteins in E.coli is a major challenge because they often tend to develop into unsolvable and inactive proteins. They aggregate into so called  inclusion bodies which prevent expression of the protein. This problem might be avoided by fusing the gene of the foreign protein with a soluble protein called solubility tags, which  function is to enhance the solubility of the foreign protein. This report investigates whether Sterol Carrier Protein-2 (SCP-2) could function as a solubility tag. The experiment was carried out by fusing SCP-2 to two recombinant proteins, Green fluorescent protein (GFP) and a form of chloroamphenicol acetyl transferase (CATΔ9). The gene fusion was then inserted into a pET-15 vector and transformed into  the E.coli strain BL21(DE3) to be expressed. The results obtained from Western blot and PageBlue staining indicates that SCP-2 does not enhance the solubility of GFP or CATΔ9 since neither of them was expressed.  Furthermore, previous studies have shown that GFP can in fact be expressed  usingmaltose binding protein (MBP) as a solubility tag. Unfortunately, no success has been made regarding CATΔ9. In conclusion, regarding the results from this report, SCP-2 does not function as a solubility tag. However, further studies should be carried out on SCP-2 with more experiments before rejecting the possibility to use SCP-2 as a solubility tag.

Expression of foreign proteins in host organisms usually results in the development of insoluble, inactive proteins. Further, these proteins have a tendency to form aggregates termed inclusion bodies. However, the formation of inclusion bodies can be avoided by fusing the gene encoding the foreign protein to a highly soluble protein. In this report Sterol Carrier Protein-2 (SCP-2) is reviewed as a possible solubility tag. The experiment was carried out by fusing SCP-2 to one of two i nsoluble proteins, Green fluorescent protein (GFP) or a form of chloramphenicol acetyl transferase (CAT∆9). The protein fusion was then inserted into the vector pET-15b, transformed in Escherichia coli and the yield of actively expressed protein was measured. The results obtained from this study, as evaluated by PageBlue staining and  Western blot, are indicating that SCP-2 does not improve the solubility of GFP or CAT∆9. Nonetheless, the solubility of GFP has earlier been increased by fusing it to the solubility tag maltose-binding protein (MBP).  Producing more soluble forms of CAT∆9  have also been tested but without success. Therefore the conclusion drawn from this experiment is that SCP-2 does not work as a solubility tag, however more research must be performed to conclude this with certainty.