Abstract Multifunctional enzyme type 2 (MFE-2) catalyses the second and the third reactions in the eukaryotic peroxisomal β-oxidation cycle, which degrades fatty acids by removing a two-carbon unit per each cycle. In addition to the 2-enoyl-CoA hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase activities, mammalian MFE-2 has also a sterol carrier protein type 2-like (SCP-2L) domain. In contrast, yeast MFE-2 has two (3R)-hydroxyacyl-CoA dehydrogenases, one 2-enoyl-CoA hydratase 2 and no SCP-2L domain. The physiological roles of yeast (3R)-hydroxyacyl-CoA dehydrogenases (A and B) were tested by inactivating them in turn by site-directed mutagenesis and testing the complementation of Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2) with mutated variants of Sc MFE-2. Growth rates were lower for fox-2 cells expressing only a single functional domain than for those expressing the Sc MFE-2. Kinetic studies with purified Candida tropicalis MFE-2 and its mutated variants show that dehydrogenase A catalyzes the reaction more efficiently with the medium- and long-chain substrates than dehydrogenase B, which in turn is the only one active with the short chain fatty acids. The structural basis of the substrate specificity difference of these two dehydrogenases was solved by X-ray crystallography together with docking studies. Protein engineering was used to produce a stabile, homogenous recombinant protein of C. tropicalis dehydrogenases in one polypeptide. The heterodimeric structure contains the typical fold of the short-chain alcohol dehydrogenase/reductase (SDR) family. Docking studies suggest that dehydrogenase A binds medium chain-length substrates as bended, whereas short chain substrates are dislocated, because they do not reach the hydrophobic contacts needed for anchoring the substrate to the active site, but are instead attracted by L44. Dehydrogenase B has a more shallow binding pocket and thus locates the short chain-length substrates correctly for catalysis. Thus the data provide clues for structural basis of the different substrate specificities. The molecular basis of the patient mutations of MFE-2 (DBP deficiency) was studied using the recently solved crystal structures of rat (3R)-hydroxyacyl-CoA dehydrogenase, human 2-enoyl-CoA hydratase and SCP-2L. The predicted effect of the mutations on protein structure could in several cases be explained, and these data supported the conclusion that a genotype-phenotype correlation exists for DBP deficiency.

Abstract The peroxisomal multifunctional enzyme type-1 (perMFE-1) is a monomeric protein of β-oxidation possessing 2-enoyl-CoA hydratase-1, Δ3-Δ 2-enoyl-CoA isomerase, and (3S)-hydroxyacyl-CoA dehydrogenase activities. The amino-terminal part of perMFE-1 shows sequence similarity to mitochondrial 2-enoyl-CoA hydratases (ECH-1) and Δ3-Δ 2-enoyl-CoA isomerases, and belongs to the hydratase/isomerase superfamily. Family members with known structures are either homotrimers or homohexamers. The purpose of this work was to elucidate the structure-function relationship of the rat perMFE-1 with special reference to the hydratase/isomerase fold. The structural adaptations required for binding of a long chain fatty acyl-CoA were studied with rat ECH-1 via co-crystallization with octanoyl-CoA. The crystal structure revealed that the long chain fatty acyl-CoA is bound in an extended conformation. This is possible because, a flexible loop moves aside and opens a tunnel, which traverses the subunit from the solvent space to the intertrimer space. Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by ECH-1. In the present work the enzymological properties of Glu144Ala and Glu164Ala variants of ECH-1 were studied. The catalytic activity of hydration was reduced about 2000-fold. It was also demonstrated that rat ECH-1 is capable of catalyzing isomerization. The replacement of Glu164 with alanine reduced the isomerase activity 1000-fold, confirming the role of Glu164 in both the hydratase and isomerase reactions. The structural factors favoring the hydratase over the isomerase reaction were addressed studying the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. These mutants had similar enzymatic properties to wild type, thus the catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on interaction with the Gln162 side chain. The perMFE-1 was divided into five functional domains based on amino acid sequence comparisons with the homologous proteins with known structures. Deletion variants of perMFE-1 showed that the folding of an enzymatically active amino-terminal hydratase/isomerase domain requires stabilizing interactions from the two carboxy-terminal domains of perMFE-1. The last carboxy-terminal domain is also required for the folding of the dehydrogenase part of perMFE-1. The dehydrogenase part of perMFE-1 was crystallized.

Abstract Multifunctional enzyme type 1 (MFE-1) is a monomeric member of the hydratase/isomerase superfamily (H/I) involved in the β-oxidation of fatty acids. MFE-1 has 2-enoyl-CoA hydratase-1, Δ3-Δ2-enoyl-CoA isomerase, and several other enoyl-CoA isomerase activities at the N-terminus. The C-terminus has (3S)-hydroxyacyl-CoA dehydrogenase activity. MFE-1 can also convert certain hydroxylated C27 bile acid synthesis intermediates. In these studies, a domain assignment of MFE-1 by sequence alignment with the H/I family (domains A and B in MFE-1) and mitochondrial monofunctional 3-hydroxyacyl-CoA dehydrogenases (HAD, domains C, D and E) was proposed. This was further improved with the structural information obtained from the crystal structure of the construct containing domains B, C, D and E (MFE1-DH). The structure of MFE1-DH resembles the bilobal structure of the α-subunit of the bacterial fatty acid metabolising complex and the mammalian HAD enzyme. The N-terminal linker helix of MFE1-DH (domain B) corresponds to helix-10 of the hydratase/isomerase enzymes having residues important for substrate contacts. Domain C adopts the classical Rossmann fold and forms the first lobe of the MFE1-DH structure. The C-terminal domains D and E form the second lobe and have local symmetry between each other. This local symmetry corresponds to the D domain-mediated dimerisation of the HAD dimer. The domain deletion studies showed that the presence of domains D and E, but not domain C, was essential to obtain a functional hydratase 1 enzyme; this can be understood from stabilising contacts from domain E to the linker helix, as seen in the MFE1-DH structure. The structure of human ACBP from liver was determined with and without a physiological ligand. This structure adopts the classical four-helix bundle of the ACBP family. The ligand binding mode seen in the presence of myristoyl-CoA shows that one ligand molecule is bound jointly by the two protein molecules of the asymmetric unit such that the fatty acid tail is bound by one protein molecule, and the 3'-phosphate AMP moiety of the CoA is bound by the other protein molecule, essentially as in known complexed ACBP structures in the monomeric binding mode. The observed ligand binding mode suggests a new model for the ACBP-mediated ligand transfer observed in biochemical in vitro studies. / Tiivistelmä Tyypin 1 monitoiminen entsyymi (MFE-1) on hydrataasi/isomeraasiperheen (H/I) jäsen ja se osallistuu rasvahappojen β-oksidaatioon. MFE-1:n N-päädyssä on 2-enoyyli-CoA-hydrataasi 1- ja Δ3-Δ2-enoyyli-CoA-isomeraasiaktiivisuus sekä useita muita enoyyli-CoA-isomeraasiaktiivisuuksia. C-päädyssä on (3S)-hydroksiasyyli-CoA-dehydrogenaasiaktiivisuus. MFE-1 voi myös katalysoida tiettyjen hydroksyloitujen C27-sappihapposynteesin välituotteiden reaktioita. Tässä tutkimuksessa määritettiin MFE-1:n domeenirakenne H/I-perheen (MFE-1:n domeenit A ja B) ja 3-hydroksiasyyli-CoA-dehydrogenaasiperheen (HAD, domeenit C, D ja E) sekvenssilinjausten perusteella. Rakennetta tarkennettiin domeenit B, C, D ja E sisältävän konstruktin (MFE1-DH) kiderakenteesta saadun tiedon avulla. MFE1-DH:n kiderakenne muistuttaa bakteerien rasvahappoja hajottavan kompleksin α-alayksikön sekä nisäkkäiden HAD-entsyymin kahdesta alayksiköstä muodostuvaa rakennetta. MFE1-DH:n N-päädyn α-kierre vastaa H/I-entsyymien kierre-10:tä, jossa sijaitsee substraattikontaktien kannalta tärkeitä aminohappotähteitä. C-domeeni muodostaa Rossmann-laskoksen ja se on MFE1-DH:n rakenteen ensimmäinen alayksikkö. C-päädyssä sijaitsevat D- ja E-domeenit muodostavat yhdessä toisen alayksikön ja niiden välillä on symmetria, joka vastaa D-domeenien välittämää HAD-entsyymien dimerisaatiota. Domeenitutkimukset osoittivat, että D- ja E-domeenien läsnä olo oli välttämätöntä hydrataasi 1:n toiminnalle, mutta C-domeeni voitiin poistaa ja säilyttää hydrataasi 1 -aktiivisuus. Havainto voitiin selittää MFE1-DH:n rakenteen avulla, jossa nähdään stabiloivia vuorovaikutuksia E-domeenin ja N-päädyn α-kierteen välillä. Ihmisen maksan ACBP:n kiderakenne määritettiin fysiologisen ligandin kanssa sekä ilman ligandia. Tämä rakenne laskostuu ACBP-perheelle tyypilliseksi neljän kierteen nipuksi. Myristoyyli-CoA:n läsnä ollessa havaitussa ligandin sitoutumistavassa yksi ligandimolekyyli on sitoutunut kahden proteiinimolekyylin välille siten, että rasvahappo-osa sitoutuu toiseen proteiinimolekyyliin ja CoA:n 3'-fosfaatti-AMP-osa sitoutuu toiseen proteiinimolekyyliin kuten tunnetuissa monomeerisissä ACBP:n sitomistavoissa. Havaitun sitoutumisen avulla voidaan ehdottaa uutta mallia ACBP:n välittämälle ligandinsiirrolle, joka on havaittu aikaisemmin biokemiallisissa in vitro -tutkimuksissa.