Yeast Peroxisomal Multifunctional Enzyme: (3R)-Hydroxyacyl-CoA Dehydrogenase Domains A and B Are Required for Optimal Growth on Oleic Acid*

The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly16 and Gly329 of the S. cerevisiae A and B domains, corresponding to Gly16, which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aΔ) and pYE352::ScMFE-2(bΔ) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aΔbΔ) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Δ)) and mutational variants of the A and B domains (Ct MFE-2(h2ΔaΔ), Ct MFE- 2(h2ΔbΔ), andCt MFE- 2(h2ΔaΔbΔ)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.

Yeast MFE-2 has been cloned from Candida tropicalis (7) and characterized from Saccharomyces cerevisiae (2). The amino acid sequence comparison of MFE-2(s) reveals that yeast enzymes contain the two domains A and B belonging to the short chain alcohol dehydrogenase/reductase superfamily (8), whereas the mammalian MFE-2 has only one. The (3R)-HADH activities of MFE-2 have been assigned to the short chain alcohol dehydrogenase/reductase domains in both the yeast (2) and mammalian enzymes (Refs. 9 and 10 and Fig. 1). An interesting question arises from what the physiological functions of the two domains are or even whether both of them show enzymatic activities. To answer this question, wild type human and yeast MFE-2, as well as their mutated variants, were tested for complementation in vivo. Wild type and mutated dehydrogenase domains of yeast MFE-2 were overexpressed, purified, and characterized in vitro. Our data show that the domains A and B have different enzymatic properties and that both domains play a functional role in the ␤-oxidation of fatty acids in yeast peroxisomes.
Growth Curve-The strain of interest was grown at 30°C for 18 h in YPD medium or SD/uracil. Cells were harvested, washed with H 2 O, and used to inoculate 10 ml of YNO medium (0.1% yeast extract, 0.67% yeast nitrogen base with amino acids, 0.1% oleic acid, and 0.5% potassium phosphate at pH 6.0) to an initial cell density of 5 ϫ 10 5 cells/ml. Cells were then incubated with vigorous agitation (250 rpm) at 30°C. Growth was assessed by counting the cell density (cells/ml) at 24-h intervals for a period of 144 h.
Production and Purification of (3R)-Hydroxyacyl-CoA Dehydrogenase-M9ZB medium supplemented with carbenicillin (50 g/ml) and chloramphenicol (34 g/ml) was used for expression experiments. 10 ml of an overnight culture of the E. coli cells containing the plasmid pET3a::CtMFE-2(h2⌬) or its mutated variants were used to inoculate 1 liter of culture. The cells were allowed to grow at 37°C under aerobic conditions until an A 600 of 0.6 was reached. The expression of the recombinant protein was induced by addition of isopropyl-1-thio-␤-Dgalactopyranoside to a final concentration of 0.4 mM. After 2 h of induction at 33°C, the cells were harvested and washed with phosphate-buffered saline (10 mM sodium phosphate, 2 mM potassium phosphate, 140 mM NaCl, 3 mM KCl, 5 mM ␤-mercaptoethanol, pH 7.4). The pellet was stored at Ϫ70°C until use.
Bacterial cell pellet (wet weight, 5.0 g) was suspended in 50 ml of 30 mM sodium phosphate, 90 mM NaCl, 1.0 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine hydrochloride hydrate, and 0.5 mM dithiothreitol at pH 7.0 (buffer A). The bacteria were lysed by adding lysozyme (125 g/ml), and the viscosity was reduced by adding DNase (25 g/ml) and RNase (25 g/ml) in the presence of 10 mM MgCl 2 for 30 min at 30°C. The insoluble material was removed by centrifugation at 30,000 ϫ g for 45 min at 4°C. The supernatant was applied to a 2. Substrate Synthesis and Enzyme Assays-(3R)-Hydroxyacyl-CoA esters were synthesized by the mixed anhydride method (14) and purified by high pressure liquid chromatography on a reversed phase Bondapak™ C18 column (Waters, Milford, MA) applying a linear acetonitrile gradient. The assay buffer contained 50 mM Tris/HCl, pH 8.0, 50 mM KCl, 1 mol of NAD ϩ (Merck), 1 mM sodium pyruvate (Sigma), 2.5 mM MgCl 2 , 10 g (21 units) of lactate dehydrogenase, and 30 nmol of (3R)-hydroxybutyryl-CoA in 0.5 ml. The dehydrogenase reaction was started by adding the sample and monitored by formation of the magnesium complex of 3-ketobutyryl-CoA at 303 nm at 22°C.
Circular Dichroism Spectroscopy-CD spectroscopy was carried out at 22°C using a Jasco J710 spectropolarimeter. Adsorption at 280 nm was measured for all samples and used for fine adjustment of the protein concentration of the sample used for CD spectroscopy. The far-UV spectra of the proteins were measured from 200 to 250 nm in 80 mM potassium phosphate, pH 7.0, with the following instrument settings: response, 1 s; sensitivity, 100 mdeg; speed, 50 nm/min; average of 30 scans.  2 The measurement was performed in a final volume of 0.5 ml at 22°C. Kinetic data were transformed to Lineweaver-Burk plots by using the GraFit computer software (Sigma). The K m values were calculated from the slopes of the curves, and the catalytic turnover numbers (k cat ) were calculated by dividing the maximal velocities with the total amount of enzyme in the reaction.

Determination of the k cat and K m Values for Recombinant (3R)-Hydroxyacyl-CoA
Other Analyses-Protein concentrations were measured with Bio-Rad protein assay reagent, and protein samples were analyzed on 12% SDS-polyacrylamide gels.

Amino Acid Sequence Alignment of Nucleotide-binding Sites of Human and Yeast MFE-2-
The G16S variant of Hs MFE-2 has been shown to result in MFE-2 deficiency (6). Because this glycine is in the nucleotide-binding site of the dehydrogenase domain and is conserved in MFE-2 of yeast, it is expected to be a functionally important amino acid residue in MFE-2 of lower eukaryotes (Fig. 2).
Growth of Yeast Cells on Oleic Acid-When pYE352::HsMFE-2 was introduced into S. cerevisiae fox-2 cells, the transformed strain regained the ability to grow on fatty acids as a carbon source as indicated by the clear zone formation on the oleic acid plate (Fig.  3A). However, no clear zones developed if the fox-2 cells transformed with pYE352::HsMFE-2(dh⌬), which encodes the G16S mu-tation (Fig. 3A). When the enzyme activities of MFE-2 were measured from soluble extracts of transformed fox-2 cells, the hydratase 2 activity was observed in all cases, whereas the (3R)-HADH activity was abolished by the G16S mutation ( Table I). The expression of the Hs MFE-2 was also confirmed by immunoblotting of samples from these yeast extracts with antibody to rat 2-enoyl-CoA hydratase 2 (3), which recognized a 79-kDa band corresponding to the predicted molecular mass of Hs MFE-2.
To investigate the kinetics of growth, the number of cells was monitored during growth in the liquid oleic acid media over time (Fig. 3C). The growth rates of S. cerevisiae fox-2 cells transformed with either pYE352::ScMFE-2(a⌬) or pYE352:: ScMFE-2(b⌬) were about 50% of that found with pYE352:: ScMFE-2 transformed cells. In agreement with the plate assay, fox-2 or fox-2 transformed with pYE352::ScMFE-2(a⌬b⌬) did not show observable growth. To verify that S. cerevisiae MFE-2 and its variants were expressed, both 2-enoyl-CoA hydratase 2 and (3R)-HADH activities were measured in yeast cell homogenates (Table I). With the exception of the fox-2 cells, hydratase 2 activity was detected for all transformants. When compared with Sc MFE-2(h2⌬), the dehydrogenase activity was decreased for the a⌬ and b⌬ variants, and it was completely lost for the a⌬b⌬ mutant.

Expression of C. tropicalis (3R)-Hydroxyacyl-CoA Dehydrogenase and Its Mutants in E. coli-Our attempts to express and purify
Sc MFE-2(h2⌬) as a recombinant protein resulted in the enzyme undergoing inactivation within a few days. However, C. tropicalis MFE-2(h2⌬) yielded a protein stable for at least one year under the conditions stated under "Experimental Procedures." Subsequent in vitro characterizations were carried out with C. tropicalis preparation.
When pET3a::CtMFE-2(h2⌬) was expressed in E. coli BL 21plysS cells, (3R)-specific HADH activity was 23 mol ϫ min Ϫ1 ϫ mg Ϫ1 in the soluble cell extract when measured with (3R)-hydroxydecanoyl-CoA, whereas the activity was below the detection limit in the cells transformed with only the vector pET3a. The expressed Ct MFE-2(h2⌬) was purified to apparent homogeneity by using two anion exchange columns: DEAE-Sephacel and Resourse Q, followed by a cation exchange column, Resourse S, and a size exclusion Superdex 200/30 HR column (Table II). It is worth of noting that the Ct MFE-2(h2⌬) preparation is, in fact, a monofunctional (3R)-HADH, which is 2 Y.-M. Qin, unpublished observation.

FIG. 2. Alignment of nucleotidebinding site from human MFE-2 with S. cerevisiae and C. tropicalis MFE-2.
HsMFE, ScMFEa, ScMFEb, CtMFEa, and CtMFEb represent human MFE-2, S. cerevisiae MFE-2 domains A and B, and C. tropicalis MFE-2 domains A and B, respectively. Alignment was performed with CLUSTAL X program. The residue number is given on both the left and right sides of the amino acid sequence. Conserved amino acids are in bold. The mutated glycine is boxed.
an enzyme not commercially available and thus can potentially be used as a tool for investigating the metabolism of fatty acids and their derivatives. Ct MFE-2(h2⌬a⌬), Ct MFE-2(h2⌬b⌬), and Ct MFE-2(h2⌬a⌬b⌬) were each expressed and purified following the same protocol, yielding apparent homogeneities (SDS-polyacrylamide gel electrophoresis analysis; Fig. 4). In immunoblotting, the antibody to Ct MFE-2 (15) recognizes these polypeptides of 66,000 Da, which agree with the molecular mass of 66,379 Da calculated from the amino acid sequence (data not shown). Native molecular masses of the proteins were determined to be 140 kDa by size exclusion chromatography (Superdex 200), indicating that they are dimers (Fig. 4). CD spectra in the far-UV region (200 -250 nm) were practically identical for both Ct MFE-2(h2⌬) and its variants (Fig. 5).
Kinetic Constants of Wild Type and Mutant Dehydrogenases-Kinetic parameters were determined for the purified Ct MFE-2(h2⌬) and its mutated variants (Table III). The Ct MFE-2(h2⌬) showed the highest catalytic efficiency (k cat /K m ) with the substrate (3R)-hydroxydecanoyl-CoA (C10). The K m value was lowest for the C10 substrate, being approximately one-fifth and one-tenth of the value of the C16 and C4 substrates, respectively. Interestingly, the (3R)-HADH activity of Ct MFE-2(h2⌬) broke into two different profiles when the mutated variants were analyzed (Table III). For Ct MFE-2(h2⌬a⌬), the catalytic constant (k cat ) of C4 was the same as for Ct MFE-2(h2⌬) (29 Ϯ 1 s Ϫ1 versus 31 Ϯ 2 s Ϫ1 ), whereas that of Ct MFE-2(h2⌬b⌬) was below the detection limit, indicating that domain B is solely responsible for the utilization of C4 substrate. The k cat values of Ct MFE-2(h2⌬a⌬) for C10 and C16 were 17 Ϯ 1 s Ϫ1 and 12 Ϯ 2 s Ϫ1 . Interestingly, for Ct MFE-2(h2⌬b⌬), the k cat values were 33 Ϯ 2 s Ϫ1 and 36 Ϯ 6 s Ϫ1 , suggesting that domain A contributes more than domain B in the metabolism of medium and long chain substrates. The activity of Ct MFE-2(h2⌬a⌬b⌬) toward the substrates tested was below the detection limit of the assays used.

DISCUSSION
The amino acid sequence of yeast peroxisomal MFE-2 reveals that the polypeptide contains two domains sharing 40% amino acid identity, both within the N-terminal half of the protein.
The domains are about 300 amino acid residues long (Fig. 1), and each of the domains contains a binding site for NAD ϩ (Rossmann fold) close to their N termini. At about 120 amino acid residues from their C termini, the domains also contain the motif Tyr-Xaa-Xaa-Xaa-Lys. These features are characteristic of the members of the short chain alcohol dehydrogenase/ reductase superfamily (8). A previous in vitro experiment with a truncated version of S. cerevisiae MFE-2 (deletion of C-terminal 271 amino acid residues) indicated that the N-terminal domains were responsible for the (3R)-HADH activity in MFE-2 (2). The present work, applying site-directed mutagen-esis to dissect the putative nucleotide-binding sites of domains A and B, shows that the growth rates of fox-2 cells transformed with either pYE352::ScMFE-2(a⌬) or pYE352::ScMFE-2(b⌬) are slower than those cells transformed with pYE352:: ScMFE-2. This suggests that domains A and B are important for utilization of fatty acids as a carbon source. The fact that the activity of hydratase 2 was not changed in the yeast extracts when measured with the C10 substrate indicates that the growth rates were not due to differences in the levels of MFE-2 expression.
The in vitro kinetic experiments with the purified recombinant yeast enzymes demonstrate that domain A is active toward medium and long chain (3R)-hydroxyacyl-CoA, whereas the B domain shows the highest catalytic rate with short chain (C4) substrates. CD far-UV spectroscopy revealed that the mo-  lar ellipticities of the proteins are similar, indicating that the mutations do not cause a change in the composition of secondary structure elements. These data, together with the observation that Ct MFE-2(h2⌬) and its variants are dimeric proteins, strongly argues that the observed differences in kinetics were due to the mutations affecting catalytic amino acid residues but not due to a change in overall folding itself. The estimated K m values for domains A and B are between 5 and 50 M, values that are within the same magnitude generally found for other ␤-oxidation enzymes (16). An interesting observation is that the sums of the k cat values for Ct MFE-2(h2⌬a⌬) and Ct MFE-2(h2⌬b⌬) are very close to k cat determined for Ct MFE-2(h2⌬).
According to the current literature, the physiological role of mammalian MFE-2 is to participate in metabolism of ␣-methyl-CoA esters (5). This is supported by the observation that ␣-methyl branched fatty acid intermediates accumulate in patients with MFE-2 deficiency (6,17). However, an accumulation of straight very long chain fatty acids has also been reported in these patients (18). In addition, in vitro mammalian MFE-2 is capable of catalyzing the cleavage of straight chain (3R)-hydroxyacyl-CoAs (3,9,19). In line with these findings, pYE352::HsMFE-2 complements fox-2 cells allowing them to grow on oleic acid plates, suggesting that mammalian MFE-2 can also potentially participate in the ␤-oxidation of straight chain substrates in vivo .
An acquisition of two domains within a single polypeptide with different chain length specificities is a novel strategy among lipid binding proteins to overcome the problems related to the metabolism of a large variety of substrates. Previously described strategies include gene duplication, which lead to the evolvement of separate enzymes, such as mammalian acyl-CoA dehydrogenases, which are presented as several paralogues (20). Adaptation can also occur via the development of adaptive substrate binding pocket, as is formed in both 2-enoyl-CoA hydratase 1 (crotonase) and the acyl-CoA binding protein. Mitochondrial hydratase 1, which catalyzes hydration reactions with trans-2-enoyl-CoA substrates from C4 to C20 in chain length, has an active site pocket in each of the subunits that is large enough to bind crotonyl-CoA (21). When a longer chain substrate is bound, the end of the acyl moiety clashes a flexible loop at the bottom of the pocket and the loop moves aside, resulting in the formation of tunnel traversing the whole subunit (22). The acyl-CoA binding protein binds the acyl-CoA  ester in a bent orientation anchoring the acyl chain in a hydrophobic cleft between two ␣-helices (23). The cleft in the bovine protein is large enough to engulf a carbon chain of up to C22 long.
The data of the experiments of yeast growing in liquid medium indicate that both domains A and B are required for optimal growth of yeast cells on fatty acid as the sole carbon source. Yeast peroxisomal MFE-2 provides an intriguing example of one polypeptide that has acquired two enzymatically active dehydrogenase domains with different chain length specificities.