Mechanistic analysis of carbon–carbon bond formation by deoxypodophyllotoxin synthase

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate–dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS’s catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel–Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.

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Structure of xyloglucan xylosyltransferase 1 reveals simple steric rules that define biological patterns of xyloglucan polymers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801105115 ◽

2018 ◽

Vol 115 (23) ◽

pp. 6064-6069 ◽

Cited By ~ 11

Author(s):

Alan T. Culbertson ◽

Jacqueline J. Ehrlich ◽

Jun-Yong Choe ◽

Richard B. Honzatko ◽

Olga A. Zabotina

Keyword(s):

Plant Cell Wall ◽

Catalytic Mechanism ◽

Industrial Applications ◽

Fold Family ◽

Glucan Chain ◽

Binding Cleft ◽

Vitro Analysis ◽

Biological Patterns ◽

In Vitro Analysis

The plant cell wall is primarily a polysaccharide mesh of the most abundant biopolymers on earth. Although one of the richest sources of biorenewable materials, the biosynthesis of the plant polysaccharides is poorly understood. Structures of many essential plant glycosyltransferases are unknown and suitable substrates are often unavailable for in vitro analysis. The dearth of such information impedes the development of plants better suited for industrial applications. Presented here are structures of Arabidopsis xyloglucan xylosyltransferase 1 (XXT1) without ligands and in complexes with UDP and cellohexaose. XXT1 initiates side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis. XXT1, a homodimer and member of the GT-A fold family of glycosyltransferases, binds UDP analogously to other GT-A fold enzymes. Structures here and the properties of mutant XXT1s are consistent with a SNi-like catalytic mechanism. Distinct from other systems is the recognition of cellohexaose by way of an extended cleft. The XXT1 dimer alone cannot produce xylosylation patterns observed for native xyloglucans because of steric constraints imposed by the acceptor binding cleft. Homology modeling of XXT2 and XXT5, the other two xylosyltransferases involved in xyloglucan biosynthesis, reveals a structurally altered cleft in XXT5 that could accommodate a partially xylosylated glucan chain produced by XXT1 and/or XXT2. An assembly of the three XXTs can produce the xylosylation patterns of native xyloglucans, suggesting the involvement of an organized multienzyme complex in the xyloglucan biosynthesis.

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The native structure of the eukaryotic ribosome

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075890 ◽

1983 ◽

Vol 41 ◽

pp. 432-433

Author(s):

R.A. Milligan ◽

P.N.T. Unwin

Keyword(s):

Ribosomal Proteins ◽

Crystal Form ◽

Native Structure ◽

X Ray Diffraction ◽

Eukaryotic Ribosome ◽

Vitro Analysis ◽

In Vitro Analysis ◽

Resolution Structure ◽

Stable Unit

A detailed understanding of the mechanism of protein synthesis will ultimately depend on knowledge of the native structure of the ribosome. Towards this end we have investigated the low resolution structure of the eukaryotic ribosome embedded in frozen buffer, making use of a system in which the ribosomes crystallize naturally.The ribosomes in the cells of early chicken embryos form crystalline arrays when the embryos are cooled at 4°C. We have developed methods to isolate the stable unit of these arrays, the ribosome tetramer, and have determined conditions for the growth of two-dimensional crystals in vitro, Analysis of the proteins in the crystals by 2-D gel electrophoresis demonstrates the presence of all ribosomal proteins normally found in polysomes. There are in addition, four proteins which may facilitate crystallization. The crystals are built from two oppositely facing P4 layers and the predominant crystal form, accounting for >80% of the crystals, has the tetragonal space group P4212, X-ray diffraction of crystal pellets demonstrates that crystalline order extends to ~ 60Å.

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1163: Radial Dilation of Ureteral Balloons: A Comparative in-Vitro Analysis

The Journal of Urology ◽

10.1016/s0022-5347(18)35308-4 ◽

2005 ◽

Vol 173 (4S) ◽

pp. 315-316

Author(s):

Kari Hendlin ◽

Brynn Lund ◽

Manoj Monga

Keyword(s):

Vitro Analysis ◽

In Vitro Analysis

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An In Vitro Analysis of Ligament Reconstruction or Extension Osteotomy on Trapeziometacarpal Joint Stability and Contact Area

Yearbook of Hand and Upper Limb Surgery ◽

10.1016/s1551-7977(08)70357-1 ◽

2007 ◽

Vol 2007 ◽

pp. 214-215 ◽

Cited By ~ 1

Author(s):

B. Wilhelmi

Keyword(s):

Contact Area ◽

Ligament Reconstruction ◽

Joint Stability ◽

Trapeziometacarpal Joint ◽

Vitro Analysis ◽

In Vitro Analysis

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Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614605 ◽

1999 ◽

Vol 81 (06) ◽

pp. 951-956 ◽

Cited By ~ 59

Author(s):

J. Corral ◽

R. González-Conejero ◽

J. Rivera ◽

F. Ortuño ◽

P. Aparicio ◽

...

Keyword(s):

Venous Thrombosis ◽

Cell Types ◽

Receptor Expression ◽

Case Control Studies ◽

Platelet Surface ◽

Vitro Analysis ◽

And Function ◽

In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

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An In Vitro Analysis of Sodium Hypochlorite Decontamination for the Reuse of Implant Healing Abutments

Journal of Oral Implantology ◽

10.1563/aaid-joi-d-19-00273 ◽

2020 ◽

Author(s):

Ahmad Almehmadi

Keyword(s):

Sodium Hypochlorite ◽

Bacterial Culture ◽

Brain Heart Infusion ◽

In Vitro Study ◽

Vitro Study ◽

Sem Images ◽

Streptococcus Sanguis ◽

Implant Dentistry ◽

Vitro Analysis

Abstract The re-use of healing abutments (HAs) has become common practice in implant dentistry for economic concerns and the aim of this in-vitro study was to assess the effect of sodium hypochlorite (NaOCl) in decontamination of HAs. 122 HAs (Used and sterilized n=107; New n=15) were procured from 3 centers, of which 3 samples were discarded due to perforation in sterilization pouch. For sterility assessment, the used HAs (n=80) were cultured in Brain Heart Infusion Broth (BHI) and Potato Dextrose Agar (PDA), bacterial isolates were identified in 7 samples. Also, 24 used HAs were stained with Phloxine B, photographed and compared to new HAs (n=5). Scanning electron microscope (SEM) assessed the differences between the two sets of HAs, following which the 7 contaminated HAs along with 24 used HAs from staining experiment (Total=31) were subsequently treated with sodium hypochlorite (NaOCl) and SEM images were observed. About 8.75% of HAs tested positive in bacterial culture; Streptococcus sanguis, Dermabacter hominis, Staphylococcus haemolyticus, and Aspergillus species were isolated. Phloxine B staining was positive for used and sterilized HAs when compared to controls. The SEM images revealed deposits in the used HAs and although treatment with NaOCl eliminated the contamination of cultured HAs, the SEM showed visible debris in the HA thread region. This in-vitro study concluded that SEM images showed debris in used HAs at screw-hole and thread regions even though they tested negative in bacterial culture. The treatment with NaOCl of used HAs showed no bacterial contamination but the debris was observed in SEM images. Future studies on the chemical composition, biological implications, and clinical influence is warranted before considering the reuse of HAs.

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