1,720,981 research outputs found
Studies on the peroxisomal multifunctional enzyme type-1 : Domain structure with special reference to the hydratase/isomerase fold
Get PDFAbstractThe peroxisomal multifunctional enzyme type-1 (perMFE-1) is a monomeric protein of β-oxidation possessing 2-enoyl-CoA hydratase-1, Δ³-Δ²-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-Δ²-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.Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Kajaaninsali (Auditorium L6), Linnanmaa, on December 17th, 2001, at 10 a.m.Abstract
The peroxisomal multifunctional enzyme type-1 (perMFE-1) is a monomeric protein of β-oxidation possessing 2-enoyl-CoA hydratase-1, Δ³-Δ²-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-Δ²-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
Properties of mouse embryonic fibroblasts deficient for uPAR/LRP1 expression and expression of USF2 from E. coli
No full textProduction and structural characterization of Mus Musculus perlecan using CyDisCo technology
No full text
Characterization of Acidovorax delafieldii PBS(A) depolymerase polyester hydrolysis activity
No full text
The role of Hydrogen Bond Network (HBNs) for the function of the oxyanion holes (OAH1 and OAH2) of the Zoogloea ramigera thiolase active site
No full text
Towards the discovery of chemical probes for diphtheria toxin-like human adenosine diphosphate ribosyltransferase 3
Get PDFDiphtheria toxin-like human ADP-ribosyltransferase 3 (ARTD3) is a poorly characterized member of the ARTD superfamily of enzymes. ARTDs are enzymes that catalyze ADP-ribosylation, a reversible post-translational modification whereby growing chains of ADP-ribose is attached onto target proteins. ARTDs are involved in a wide variety of roles within the cell, including DNA damage repair and maintenance of genomic stability. ARTD3 is activated by damaged DNA and believed to play a role in double-strand break repair (DSBR) in the non-homologous end-joining (NHEJ) pathway, and has been implicated as a drug target in the treatment of cancer. In this study, an activity assay previously reported for other ARTDs was adopted for ARTD3 in order to screen the enzyme with two compound libraries consisting of 918 compounds in order to discover new potential chemical probes for the protein. The assay was validated through statistical criteria and its performance tested in a preliminary screen with 32 known ARTD inhibitors and their analogs. From all the compounds screened, 12 were ordered and 8 verified as hits in a counter-screen at 10 µM compound concentration. These 8 compounds were then further characterized with thermofluor to determine their ability to bind ARTD3 catalytic domain, fluorescence polarization (FP) to determine their effects on ARTD3’s binding affinity with DNA and the compounds had their IC50 values were measured. 6 compounds that exhibited interesting results in these experiments were classified according to their proposed mechanism of inhibition or compound class. These compounds consisted of two DNA chelating topoisomerase inhibitors, one insecticide, one heavy-metal containing polycyclic hydrocarbon (PHC) and two unclassified tumor suppressors. None of the compounds stabilized full-length ARTD3 or the catalytic fragment, but the DNA chelating agents lowered the binding affinity of ARTD3 to DNA. While no traditional ARTD inhibitor with potential for use as a chemical probe was discovered, the effects of DNA chelating agents on ARTD3’s binding affinity with DNA could be further investigated via binding affinity studies such as isothermal titration calorimetry (ITC). One of the unclassified tumor suppressors with decent potency (IC50: 2.7 µM) could also be tested against other ARTDs and if it was found to be selective against ARTD3, its binding to ARTD3 could be determined with co-crystallization and x-ray crystallography
- …
