Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF

AbstractNon-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

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Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF

10.21203/rs.3.rs-736558/v1 ◽

2021 ◽

Author(s):

Hui Tao ◽

Takahiro Mori ◽

Heping Chen ◽

Shuang Lyu ◽

Akihito Nonoyama ◽

...

Keyword(s):

High Efficiency ◽

Cyclopropane Ring ◽

Heme Iron ◽

Oxidation Reactions ◽

Loop Region ◽

Non Heme Iron ◽

Vitro Analysis ◽

Continuous Oxidation ◽

In Vitro Analysis

Abstract Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis reveals that SptF catalyzes four continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced novel cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF reveal the molecular basis of the enzyme reactions, and suggest that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

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Comparison of urea clearance in low-efficiency low-flux vs. high-efficiency high-flux dialyzer membrane with reduced blood and dialysate flow: An in vitro analysis

Hemodialysis International ◽

10.1111/hdi.12054 ◽

2013 ◽

Vol 18 (1) ◽

pp. 172-174 ◽

Cited By ~ 5

Author(s):

Raj Munshi ◽

Suhail Ahmad

Keyword(s):

High Efficiency ◽

High Flux ◽

Urea Clearance ◽

Dialysate Flow ◽

Low Efficiency ◽

Vitro Analysis ◽

In Vitro Analysis

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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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