Supramolecular Chemistry of Cyclodextrins in Enzyme Technology

Villalonga, Reynaldo; Cao, Roberto; Fragoso, Alex

doi:10.1021/cr050253g

Cited by 355 publications

(189 citation statements)

References 301 publications

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“…VD 3 and 25(OH)VD 3 were dissolved in dimethyl sulfoxide at concentrations ranging from 50 M to 2 mM, and 10 l of the substrate solution was added to 990 l of reaction mixture containing 2.5 M Vdh-WT or Vdh mutants, 20 mM Tris-HCl, pH 7.5, and 0.05% PM␤CD or 0.02% ␥-cyclodextrin (␥CD). PM␤CD and ␥CD were used to increase the water solubility of VD 3 and 25(OH)VD 3 , respectively (9,23). CD concentrations referred to those adopted in actual VD 3 bioconversion by P. autotrophica.…”

Section: Methodsmentioning

confidence: 99%

Structural Evidence for Enhancement of Sequential Vitamin D3 Hydroxylation Activities by Directed Evolution of Cytochrome P450 Vitamin D3 Hydroxylase

Yasutake

Fujii

Nishioka

et al. 2010

Journal of Biological Chemistry

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Vitamin D 3 hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D 3 (VD 3 ) to 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 VD 3 ) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD 3 , i.e. the conversion of VD 3 to 25-hydroxyvitamin D 3 (25(OH)VD 3 ) and then of 25(OH)VD 3 to 1␣,25(OH) 2 VD 3 , a hormonal form of VD 3 . Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD 3 -bound, and 25(OH)VD 3 -bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substratebound structure of Vdh-K1 accommodates both VD 3 and 25(OH)VD 3 but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD 3 hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD 3 25-hydroxylase and 25(OH)VD 3 1␣-hydroxylase activities.3 is a B-ring opening secosteroid involved in a wide variety of biological functions in mammals (1). In humans, VD 3 is converted into its physiologically active form, 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 VD 3 ), via hydroxylation reactions that are catalyzed by several cytochrome P450s (CYPs) (1, 2). The first hydroxylation is done at the C25 position of VD 3 by CYP27A1 (2, 3) and CYP2R1 (2, 4) in the liver to produce 25-hydroxyvitamin D 3 (25(OH)VD 3 ). The second proceeds at the C1␣ position of 25(OH)VD 3 by CYP27B1 in the kidney (5) (Fig. 1). The final product, 1␣,25(OH) 2 VD 3 , functions as a hormone with a critical role in maintaining calcium and phosphate homeostasis as well as in controlling the differentiation and proliferation of multiple cell types (1, 2, 6). Indeed, the many symptoms associated with VD 3 deficiency and the VD metabolic disorder, which include psoriasis, osteoporosis, rickets, and hypoparathyroidism, are treated using 1␣,25(OH) 2 VD 3 and its derivatives (1).Although the chemical synthesis of 1␣,25(OH) 2 VD 3 from cholesterol is an established method, it is inefficient, the maximum yield is no more than 1% (7). Alternatively, biocatalytic conversion by the actinomycete Pseudonocardia autotrophica is currently in practical use for the industrial production of 1␣,25(OH) 2 VD 3 (8, 9). We have recently cloned the gene encoding the VD 3 hydroxy...

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Section: Methodsmentioning

confidence: 99%

Structural Evidence for Enhancement of Sequential Vitamin D3 Hydroxylation Activities by Directed Evolution of Cytochrome P450 Vitamin D3 Hydroxylase

Yasutake

Fujii

Nishioka

et al. 2010

Journal of Biological Chemistry

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“…These types of compounds deprotonate upon self-assembled monolayers (SAMs), a process that neutralizes the negatively charged sulfur-containing compound assuming the formation of gold (4,5). SAMs of alkane thiols http://bmbreports.org on gold containing a functional terminal group have served to associate additional monolayers of enzymes by covalent conjugation, supramolecular association, physical adsorption and electrostatic interactions (6)(7)(8)(9)(10)(11). Previous study found that immobilization of enzyme onto gold nanoparticle's surfaces, without any surface modification, provided colloidal stability, which result to significantly enhanced catalytic activity (11).…”

Section: Gold Nanoparticlesmentioning

confidence: 99%

Inorganic nanomaterial-based biocatalysts

Lee

Lee²,

Chang³

et al. 2011

BMB Reports

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Over the years, nanostructures have been developed to enable to support enzyme usability to obtain highly selective and efficient biocatalysts for catalyzing processes under various conditions. This review summarizes recent developments in the nanostructures for enzyme supporters, typically those formed with various inorganic materials. To improve enzyme attachment, the surface of nanomaterials is properly modified to express specific functional groups. Various materials and nanostructures can be applied to improve both enzyme activity and stability. The merits of the incorporation of enzymes in inorganic nanomaterials and unprecedented opportunities for enhanced enzyme properties are discussed.

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“…The commercially available cyclomaltooligosaccharides (cyclodextrins; CDs) appear particularly attractive on those grounds (15)(16). CDs combine biocompatibility, availability, and a tubular symmetric framework with well differentiated faces that can be selectively modified in order to attain an exquisite control of the presentation and orientation of the assembled elements (17)(18)(19).…”

Section: Introductionmentioning

confidence: 99%

Insights in cellular uptake mechanisms of pDNA–polycationic amphiphilic cyclodextrin nanoparticles (CDplexes)

Díaz‐Moscoso

Vercauteren

Rejman

et al. 2010

Journal of Controlled Release

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have equally contributed to the reported work. Abstr actIt is generally recognized that the major obstacle to efficient gene delivery is cellular internalization and endosomal escape of the DNA. Recently, we have developed a modular strategy for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) capable of complexing and compacting DNA into homogeneous nanoparticles (<70 nm).Since paCDs resemble both cationic polymers and cationic lipids, it is conceivable that the corresponding pDNA-paCD nanoparticles (CDplexes) might use the cell uptake and endosomal escape mechanisms described for both lipoplexes and polyplexes. To verify this hypothesis, we have now investigated the uptake and transfection efficiencies of CDplexes in the presence of several inhibitors of endocytosis, namely chlorpromazine, genistein, dynasore and methylated β-cyclodextrin (MbCD). Our data show that CDplexes obtained from paCD 1, which ranks among the most efficient paCD gene vectors reported up to date, are internalized by both clathrin-dependent (CDE) and clathrin-independent endocytosis (CIE), both processes being cholesterol-and dynamin-dependent. We observed that the largest fraction of gene complexes is taken up via CDE, but this fraction is less relevant for transfection. The smaller fraction that is internalized via the CIE pathway is predominantly responsible for successful transfection. Intr oductionThe successful delivery of therapeutic genes into cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. During the last decade, under the advent of nanotechnology, a broad diversity of creative materials featuring promising properties for nonviral gene delivery applications has emerged, (1). Cationic polymers and lipids, or their combinations, have shown the ability to bind nucleic acids through electrostatic interactions and condense them into complexes that can be readily internalized by cells (2-3). The gene delivering capabilities of many of these systems have been thoroughly investigated. Yet, drawing firm conclusions that might serve to provide feedback for the design of improved delivery entities is a delicate issue as a consequence of the essentially polydisperse nature of such structures (4).The control of the architecture of multifunctional macromolecules is a major determinant in the rational design of successful nonviral gene delivery systems. Novel 3 strategies have been implemented to build up well defined constructs featuring selfassembling capabilities in the presence of nucleic acids. In this context, the unique characteristics of dendrimers and other highly ordered clusters, such as uniformity, monodispersity and multivalency, have attracted increasing attention (5-8). Homogeneous external functionalization of these platforms becomes, however, exponentially more complicated as the dendrimer generation increases (9-10). The use of preorganized macrocyclic scaffolds, such as calixarenes, to achieve a precise alignment of functiona...

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Supramolecular Chemistry of Cyclodextrins in Enzyme Technology

Cited by 355 publications

References 301 publications

Structural Evidence for Enhancement of Sequential Vitamin D3 Hydroxylation Activities by Directed Evolution of Cytochrome P450 Vitamin D3 Hydroxylase

Structural Evidence for Enhancement of Sequential Vitamin D3 Hydroxylation Activities by Directed Evolution of Cytochrome P450 Vitamin D3 Hydroxylase

Inorganic nanomaterial-based biocatalysts

Insights in cellular uptake mechanisms of pDNA–polycationic amphiphilic cyclodextrin nanoparticles (CDplexes)

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