Evidence for Domain Structures of the Trifunctional Protein and the Tetrafunctional Protein Acting in Glyoxysomal Fatty Acid β‐Oxidation

Abstract

In plant glyoxysomes, an enzyme activity responsible for a particular step in the fatty acid , ! loxidation is located on more than one protein species. Various monofunctional enzymes and two forms of a multifunctional protein are involved in the degradation of cis-unsaturated fatty acids. A',A*-Enoyl-CoA isomerase activity, previously found to be located on a monofunctional dimeric protein, is attributable to one form of the monomeric multifunctional protein (MFP). The presence or absence of isomerase activity allows us to differentiate between the tetrafunctional 76.5-kDa isoform (MFP 11) and the trifunctional 74-kDa isoform (MFP I) in cucumber (Cucumis sativus) cotyledons. Both MFP I and MFP I1 exhibited blocked N-terminal structures. MFP I and MFP I1 are distinguishable from each other by their susceptibility to limited proteolysis. A series of examples is presented describing the preparation of enzymically active proteolytic fragments. We demonstrate that both forms of the monomeric MFP are composed of domains separable from each other without loss of activity. By fragmentation of MFP I and subsequent chromatography, a 60-kDa peptide was purified retaining hydratase and epimerase activity but lacking dehydrogenase activity. In addition, a highly positively charged fragment was observed carrying solely dehydrogenase activity. From MFP 11, a 36-kDa fragment with hydratase activity was characterized. An enzymically inactive 46-kDa fragment was prepared from MFP I1 and sequenced at its unblocked N-terminus.Comprehensive studies of the enzymes from glyoxysomes and peroxisomes have provided evidence for a close phylogenetic relationship [ 1 -41. For fatty acid P-oxidation, all forms of microbodies possess a multifunctional protein (MFP) as marker protein [4-71. Studies indicate that this protein represents a fusion product of two, three or four single enzyme peptides connected in a linear sequence [5,8, 91. This leads to the assumption that several domains as compact globular structures form the tertiary structures of MFP. Portions of the polypeptide chain may fold to stable tertiary structures, independent of the presence of other parts of the polypeptide chain. Domains of a large protein like MFP may be connected by short flexible regions free of enzymic activity. If such flexible regions, unlike the compact globular structure bearing active sites, are easily accessible for proteases, we expected that domains with single active centers can be cleaved off, purified and analyzed. It is suggestive that at least one exposed hinge region exists in the MFP, namely between the 3-hydroxyacyl-CoA dehydrogenase region and a region bearing, among others, the 2-enoyl-CoA hydratase activity. In this context, we consider the two forms of MFP with different size and different number of functions as model systems for demonstrating the domain structure of proteins.Correspondence to H. Kindl, FB Chemie, Philipps-Universitat, D-35032 Marburg, GermanyEnzymes. L-3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) ; d3,dZ-enoyl-CoA iso...

The multifunctional protein At MFP2 is co-ordinately expressed with other genes of fatty acid β-oxidation during seed germination in Arabidopsis thaliana (L.) Heynh

“…This difference in activities is broadly consistent with that reported in cucumber [23]. T o our knowledge this is the first report describing the expression of a MFP gene in plants.…”

“…M F P I, I1 and I11 are associated with germinating seedlings whereas M F P IV is a leaf peroxisomal form [23]. Unlike ACOX these isozymes share broad overlapping substrate specificities [23]. A full-length cDNA (MFP-a; GenBank accession no.…”

“…Crotonyl-CoA and hydroxybutyryl-CoA were used respectively as substrates since cucumber MFPs show a high level of activity with these compounds [23]. Both the 2-trans-enoyl-CoA hydratase and L-3hydroxyacyl-CoA dehydrogenase activities peaked 2-3 days after imbibition and declined to basal levels as the seedling developed (Figure 3 A).…”

Metabolism of Ferulic Acid to Vanillin

“…Multifunctional fatty acid oxidation proteins of the enoylSCoA hydratase superfamily, including the mitochondrial (36,37), peroxisomal (38,39) and plant glyoxysomal (40,41) multifunctional proteins, the large ␣-subunit encoded by the fadB gene of E. coli (24,42), and the corresponding protein encoded by the fao gene of P. fragi (25), all possess L-3-hydroxyacyl dehydrogenase activity. This is responsible for the ␤-oxidation reaction to the corresponding 3-oxo acid.…”

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

“…These are acyl-CoA oxidase, multifunctional protein, and thiolase. The catalytic and molecular properties of the two former components have been studied intensively (Frevert and Kindl 1980a;Miernyk and Trelease 1981;Gerhardt 1985;Kirsch et al 1986;Behrends et al 1988;GuÈ hnemann-SchaÈ fer et al 1994;Preisig-MuÈ ller et al 1994; GuÈ hnemann-SchaÈ fer and Kindl 1995;Hooks et al 1996;Hayashi et al 1998Hayashi et al , 1999. Available information on the glyoxysomal thiolase predominantly refers to the isolation of cDNA clones encoding the thiolase, to thiolase biosynthesis and related aspects (Preisig-MuÈ ller and Kindl 1993;Bojorquez and Gomez-Lim 1995;Olesen and Brandt 1995;Kato et al 1996;Olesen et al 1997).…”