Solvent Isotope Effects in Reactions of Human Medium-Chain Acyl-CoA Dehydrogenase Active Site Mutants

Gradinaru, Robert �Vasile; Schowen, Richard L.; Ghisla, Sandro

doi:10.1021/bi0614582

Cited by 8 publications

(5 citation statements)

References 47 publications

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“…More likely, the binding of the inhibitor might shield the active center from dioxygen, as has been observed e.g. with reduced medium-chain acyl-CoA dehydrogenase [ 46 ]. The effect of norflurazon is reversible.…”

Section: Discussionmentioning

confidence: 99%

Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis

et al. 2015

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Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FADred, while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.

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Section: Discussionmentioning

confidence: 99%

Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis

et al. 2015

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“…Finally a fit of the data (curves) obtained from the simulation and of the experimental data points is done with the application KaleidaGraph using the same mono-or bi-exponential equation (of the type: y ϭ A⅐e Ϫk1t ϩ B⅐e Ϫk2t ϩ C, where A and B are amplitudes and C the initial value). For more detailed information, refer to Gradinaru et al (26).…”

Section: Methodsmentioning

confidence: 99%

Plant Carotene Cis-Trans Isomerase CRTISO

Ghisla

Hirschberg

et al. 2011

Journal of Biological Chemistry

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The carotene cis-trans isomerase CRTISO is a constituent of the carotene desaturation pathway as evolved in cyanobacteria and prevailing in plants, in which a tetra-cis-lycopene species, termed prolycopene, is formed. CRTISO, an evolutionary descendant of the bacterial carotene desaturase CRTI, catalyzes the cis-to-trans isomerization reactions leading to all-trans-lycopene, the substrate for the subsequent lycopene cyclization to form all-trans-␣/␤-carotene. CRTISO and CRTI share a dinucleotide binding motif at the N terminus. Here we report that this site is occupied by FAD in CRTISO. The reduced form of this cofactor catalyzes a reaction not involving net redox changes. Results obtained with C(1)-and C(5)-deaza-FAD suggest mechanistic similarities with type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase (IDI-2). CRTISO, together with lycopene cyclase CRTY and IDI-2, thus represents the third enzyme in isoprenoid metabolism belonging to the class of non-redox enzymes depending on reduced flavin for activity. The regional specificity and the kinetics of the isomerization reaction were investigated in vitro using purified enzyme and biphasic liposome-based systems carrying specific cis-configured lycopene species as substrates. The reaction proceeded from cis to trans, recognizing half-sides of the symmetrical prolycopene and was accompanied by one trans-to-cis isomerization step specific for the C(5)-C(6) double bond. Rice lycopene ␤-cyclase (OsLCY-b), when additionally introduced into the biphasic in vitro system used, was found to be stereospecific for all-trans-lycopene and allowed the CRTISO reaction to proceed toward completion by modifying the thermodynamics of the overall reaction.Carotenoids belong to a large isoprenoid family, some members of which are indispensible in all photosynthetic organisms; however, they can also be formed by some heterotrophic bacteria and fungi. In photosynthesis, carotenoids play an important role in light harvesting and in the protection of cells from photo damage caused by reactive oxygen species. In addition, some carotenoids are precursors of phytohormones such as abscisic acid (1) and the strigolactones (2, 3). In human nutrition, carotenoids containing at least one unsubstituted ␤-ionone ring serve as provitamin A carotenoids (for review, see Ref. 4). In recent years, a wealth of molecular information on carotenogenesis in plants and microorganisms has become available (see Refs. 5, 6 for reviews on plant carotenogenesis), however, information on the enzymology and biochemistry of the involved reactions is comparatively scarce.In the carotenoid biosynthetic pathway, phytoene is the first carotene formed. Phytoene is a colorless C 40 triene prenyl hydrocarbon, which undergoes four desaturation steps during which double bonds are introduced to finally form the fully conjugated undecaene chromophore of the red-colored lycopene (Fig. 1). There are two divergent classes of carotene desaturases catalyzing the formation of the identical product (all-trans-lyc...

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“…This allows the estimation of the spectra of intermediates, of rate constants, and of the concentration of intermediates as a function of time. The same program was used to simulate kinetic processes [35]. Of relevance for the present case, the estimation of the lower limits of the rates of steps k 1 and k −1 was performed in two steps.…”

Section: Methodsmentioning

confidence: 99%

Optimization of d‐amino acid oxidase for low substrate concentrations – towards a cancer enzyme therapy

et al. 2009

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d‐Amino acid oxidase (DAAO) has recently become of interest as a biocatalyst for industrial applications and for therapeutic treatments. It has been used in gene‐directed enzyme prodrug therapies, in which its production of H2O2 in tumor cells can be regulated by administration of substrate. This approach is limited by the locally low O2 concentration and the high Km for this substrate. Using the directed evolution approach, one DAAO mutant was identified that has increased activity at low O2 and d‐Ala concentrations and a 10‐fold lower Km for O2. We report on the mechanism of this DAAO variant and on its cytotoxicity towards various mammalian cancer cell lines. The higher activity observed at low O2 and d‐Ala concentrations results from a combination of modifications of specific kinetic steps, each being of small magnitude. These results highlight the potential in vivo applicability of this evolved mutant DAAO for tumor therapy.

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Solvent Isotope Effects in Reactions of Human Medium-Chain Acyl-CoA Dehydrogenase Active Site Mutants

Cited by 8 publications

References 47 publications

Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis

Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis

Plant Carotene Cis-Trans Isomerase CRTISO

Optimization of d‐amino acid oxidase for low substrate concentrations – towards a cancer enzyme therapy

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