The three-dimensional structure of complex I from Yarrowia lipolytica: A highly dynamic enzyme (original) (raw)
Related papers
2011
1. Fab co-complexes of proton pumping NADH:ubiquinone oxidoreductase (complex I) Fab fragments suitable for co-crystallization with complex I were generated using an immobilized papainbased protocol. The binding of the antibody fragments to complex I was verified using Surface Plasmon Resonance and size exclusion chromatography. The binding constants of the antibodies and their respective Fab fragments were found to be in the nanomolar range. This work presents the first report on successful crystallization of complex I (proton pumping NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica with proteolytic Fab fragments. The quality of the crystals was significantly improved when compared to the initial experiments and the best crystals diffracted X-rays to a resolution of ~7 Å. The activity of complex I remained uninfluenced by antibody fragment binding. The initial diffraction data suggest that the complex I/Fab co-complex crystals represent a space group different to the one ob...
Exploring the Conformational States and Rearrangements of Yarrowia lipolytica Lipase
Biophysical Journal, 2010
We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.
Topological characterization and modeling of the 3D structure of lipase fromPseudomonas aeruginosa
FEBS Letters, 1993
Lipase from &x4domonas ueruginosu is a M, 29 kDa protein with a single functional disultide bond as shown by a shift in electrophoretic mobility after treatment with dithiothreitol and iodoacetamide. Liited proteolysis of lipase with Staphylococcus uureus protease V8 resulted in cleavage after amino acid residues Asp)' and GIu~. Comparison of the lipase amino acid sequence with those of other hydrolases with known 3D structures indicated that the folding pattern might be compatible with the al/l hydrolase fold, thereby allowing us to construct a 3D model which fitted the biochemical properties. The model predicts a catalytic triad consisting of Se?, Asp*" and Hi?', and contains a disuhide bond connecting residues Cys'S) and C~S*~~. Residues Asp3* and Gl@ are located at the surface of the enzyme, whereas the disulfide bond is rather inaccessible, which is in agreement with the finding that the protein needed to be partly unfolded before a reduction of the disultide bond could take place. A striking prediction from the model was the lack of a lid-like a-helical loop structure covering the active site which confers to other well-characterized lipases a unique property known as interfacial activation. Experimental determination of lipase activity under conditions where the substrate existed either as monomeric solutions or aggregates confirmed the absence of interfacial activation.
Immunoelectron microscopy of enzymes, multienzyme complexes, and selected other oligomeric proteins
Electron Microscopy Reviews, 1992
The collective term "immunoelectron microscopy" subsumes a number of techniques in which the biological material is decorated with specific antibodies, prior to being visualized in the electron microscope. In this article, we have reviewed literature on immunoelectron microscopy that focusses on the analysis of the molecular architecture of proteins, in particular of enzymes and of multienzyme complexes. Molecular immunoelectron microscopy has been remarkably successful with multi-subunit enzymes of complex quaternary structures, and in many cases the data have been the basis for the eventual development of detailed three-dimensional molecular models. The elucidation of subunit composition and juxtaposition of a given enzyme, an important accomplishment in itself, has in turn stimulated and guided discussions on the catalytic mechanism; illustrative examples include F. ATPase and citrate lyase, among others. Here we have chosen a variety of enzymes, multienzyme complexes, and non-enzymatic proteins to demonstrate the versatility of immunoetectron microscopy, to illustrate methodological prerequisites and limitations, and to discuss significance and implications of individual immunoelectron microscopy studies.
Crystallographic studies on membrane and cytoplasmic enzymes
2013
Pyrimidine 5'nucleotidase PF05822 159 DUF705 Protein of unknown function PF05152 68 IPR007827 particular the catalytic nucleophile Asp8 and the acid/base Asp10. Functional characterization of this enzyme revealed that it does not utilize β-glucose 1-phosphate or α-mannose 1-phosphate (Neves et al., 2006). However, this lactococcal α-phosphoglucomutase catalyzes reversible conversion of α-glucose 1-phosphate to glucose 6phosphate. From now on this α-phosphoglucomutase from Lactococcus lactis will be abbreviated as APGM.
Journal of Structural Biology, 2001
We determined the structure of the V 1 -ATPase from Manduca sexta to a resolution of 1.8 nm, which for the first time reveals internal features of the enzyme. The V 1 -ATPase consists of a headpiece of 13.5 nm in diameter, with six elongated subunits, A 3 and B 3 , of approximately equal size, and a stalk of 6 nm in length that connects V 1 with the membranebound domain, V O . At the center of the molecule is a cavity that extends throughout the length of the A 3 B 3 hexamer. Inside the cavity the central stalk can be seen connected to only two of the catalytic A subunits. The structure was obtained by a combination of the Random Conical Reconstruction Technique and angular refinements. Additional recently developed techniques that were used include methods for simultaneous translational rotational alignment of the 0°images, contrast transfer function correction for tilt images, and the Two-Step Radon Inversion Algorithm.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2015
Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium-and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.
Applied Biochemistry and Biotechnology, 2015
The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via Nterminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6×10 5 molecules per cell, which is close to the theoretical maximum (2×10 6 molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60°C for 5 min of incubation, and 83 % of original activity after incubation at 50°C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33-and 45-h reactions, respectively.
Structure, 1999
Background: Thiolases are ubiquitous and form a large family of dimeric or tetrameric enzymes with a conserved, five-layered αβαβα catalytic domain. Thiolases can function either degradatively, in the β-oxidation pathway of fatty acids, or biosynthetically. Biosynthetic thiolases catalyze the biological Claisen condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in a wide range of biosynthetic pathways, including those that generate cholesterol, steroid hormones, and various energy-storage molecules.