J K Hiltunen

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.

The isomerase and hydratase reaction mechanism of the crotonase active site of the multifunctional enzyme (type‐1), as deduced from structures of complexes with 3S‐hydroxy‐acyl‐CoA

The multifunctional enzyme, type‐1 (MFE1) is involved in several lipid metabolizing pathways. It catalyses: (a) enoyl‐CoA isomerase and (b) enoyl‐CoA hydratase (EC 4.2.1.17) reactions in its N‐terminal crotonase part, as well as (3) a 3S‐hydroxy‐acyl‐CoA dehydrogenase (HAD; EC 1.1.1.35) reaction in its C‐terminal 3S‐hydroxy‐acyl‐CoA dehydrogenase part. Crystallographic binding studies with rat peroxisomal MFE1, using unbranched and branched 2E‐enoyl‐CoA substrate molecules, show that the substrate has been hydrated by the enzyme in the crystal and that the product, 3S‐hydroxy‐acyl‐CoA, remains bound in the crotonase active site. The fatty acid tail points into an exit tunnel shaped by loop‐2. The thioester oxygen is bound in the classical oxyanion hole of the crotonase fold, stabilizing the enolate reaction intermediate. The structural data of these enzyme product complexes suggest that the catalytic base, Glu123, initiates the isomerase reaction by abstracting the C2‐proton from the substrate molecule. Subsequently, in the hydratase reaction, Glu123 completes the catalytic cycle by reprotonating the C2 atom. A catalytic water, bound between the OE1‐atoms of the two catalytic glutamates, Glu103 and Glu123, plays an important role in the enoyl‐CoA isomerase and the enoyl‐CoA hydratase reaction mechanism of MFE1. The structural variability of loop‐2 between MFE1 and its monofunctional homologues correlates with differences in the respective substrate preferences and catalytic rates.

Crystallization and characterization of the dehydrogenase domain from rat peroxisomal multifunctional enzyme type 1

Peroxisomal multifunctional enzyme type 1 from rat (perMFE-1) is a monomeric multidomain protein shown to have 2-enoyl-CoA hydratase/Delta(3)-Delta(2)-enoyl-CoA isomerase and (3S)-hydroxyacyl-CoA dehydrogenase domains followed by a C-terminal extension of 130 amino acids with unknown function apart from being a carrier of the peroxisomal targeting signal type 1. The truncated perMFE-1 without the N-terminal hydratase/isomerase domain (perMFE-1DH; residues 260-722) was overexpressed as an enzymatically active recombinant protein, purified and characterized. Using (3S)-hydroxydecanoyl-CoA as a substrate, the specific enzymatic activity of perMFE-1DH was determined to be 2.2 micromol min(-1) mg(-1), comparable with that of perMFE-1 purified from rat liver (2.8 micromol min(-1) mg(-1)). The protein was crystallized in the apo form by the hanging-drop method and a complete data set to 2.45 A resolution was collected using a rotating-anode X-ray source. The crystals have primitive tetragonal symmetry, with unit-cell parameters a = b = 125.9, c = 60.2 A.

Pedagogical basis of DAS formalism in engineering education

The paper presents a new approach for a bachelor-level curriculum structure in engineering. The approach is called DAS formalism according to its three phases: description, analysis and synthesis. Although developed specifically for process and environmental engineering, DAS formalism has a generic nature and it could also be used in other engineering fields. The motivation for this new curriculum structure originates from the urge to solve the problems that engineering education has faced during the past decades, e.g. student recruitment problems and dissatisfactory learning outcomes. The focus of this paper is on the structure of the curriculum but the content is also discussed when it has an effect on the structure and its implementation. The presented structure, i.e. DAS formalism, builds upon the ideas of some classical pedagogical theories, which have regularly been applied at course level but seldom used to solve curriculum-level issues.

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

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.

A triangular approach to integrate research, education and practice in higher engineering education

ABSTRACT Separate approaches in engineering education, research and practice are not very useful when preparing students for working life; instead, integration of education, research and industrial practices is needed. A triangular approach (TA) as a method to accomplish this integration and as a method to provide students with integrated expertise is proposed. The results from the application of TA, both at the course and programme level, indicate that the approach is suitable for developing engineering education. The student pass rate for courses where TA has been used has been higher than for previous approaches, and the student feedback has been very positive. Although TA aims to take both theoretical and practical aspects of engineering as well as research and education into account, the approach concentrates mainly on activities and therefore leaves the goals of these activities as well as the values behind these goals uncovered.