In Vitro Reconstitution and Analysis of the Chain Initiating Enzymes of the R1128 Polyketide Synthase†

Biochemistry ◽  
2001 ◽  
Vol 40 (49) ◽  
pp. 14855-14861 ◽  
Author(s):  
Eric S. Meadows ◽  
Chaitan Khosla
Keyword(s):  
Polyketide Synthase ◽  
In Vitro Reconstitution

scholarly journals In vitro reconstitution of α-pyrone ring formation in myxopyronin biosynthesis

2015 ◽  
Vol 6 (8) ◽  
pp. 5076-5085 ◽  
Author(s):  
H. Sucipto ◽  
J. H. Sahner ◽  
E. Prusov ◽  
S. C. Wenzel ◽  
R. W. Hartmann ◽  
...  
Keyword(s):  
Natural Products ◽  
Polyketide Synthase ◽  
Ring Formation ◽  
Type I ◽  
Pyrone Ring ◽  
In Vitro Reconstitution

α-Pyrone rings exist in many polyketide synthase (PKS) derived natural products. We report the first in vitro reconstitution of α-pyrone ring formation by a type I PKS using chemically synthesized substrates.

scholarly journals Partial In Vitro Reconstitution of an Orphan Polyketide Synthase Associated with Clinical Cases of Nocardiosis

2016 ◽  
Vol 11 (9) ◽  
pp. 2636-2641 ◽  
Author(s):  
James Kuo ◽  
Stephen R. Lynch ◽  
Corey W. Liu ◽  
Xirui Xiao ◽  
Chaitan Khosla
Keyword(s):  
Polyketide Synthase ◽  
In Vitro Reconstitution ◽  
Clinical Cases

scholarly journals Biosynthesis of Hexahydroxyperylenequinone Melanin via Oxidative Aryl Coupling by Cytochrome P-450 in Streptomyces griseus

2005 ◽  
Vol 187 (23) ◽  
pp. 8149-8155 ◽  
Author(s):  
Nobutaka Funa ◽  
Masanori Funabashi ◽  
Yasuo Ohnishi ◽  
Sueharu Horinouchi
Keyword(s):  
Polyketide Synthase ◽  
Uv Light ◽  
Streptomyces Griseus ◽  
Type I ◽  
Type Iii Polyketide Synthase ◽  
Melanin Biosynthesis ◽  
Type Iii ◽  
In Vitro Reconstitution ◽  
Cytochrome P 450

ABSTRACT Dihydroxyphenylalanine (DOPA) melanins formed from tyrosine by tyrosinases are found in microorganisms, plants, and animals. Most species in the soil-dwelling, gram-positive bacterial genus Streptomyces produce DOPA melanins and melanogenesis is one of the characteristics used for taxonomy. Here we report a novel melanin biosynthetic pathway involving a type III polyketide synthase (PKS), RppA, and a cytochrome P-450 enzyme, P-450mel, in Streptomyces griseus. In vitro reconstitution of the P-450mel catalyst with spinach ferredoxin-NADP+ reductase/ferredoxin revealed that it catalyzed oxidative biaryl coupling of 1,3,6,8-tetrahydroxynaphthalene (THN), which was formed from five molecules of malonyl-coenzyme A by the action of RppA to yield 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (HPQ). HPQ readily autopolymerized to generate HPQ melanin. Disruption of either the chromosomal rppA or P-450mel gene resulted in abolishment of the HPQ melanin synthesis in S. griseus and a decrease in the resistance of spores to UV-light irradiation. These findings show that THN-derived melanins are not exclusive in eukaryotic fungal genera but an analogous pathway is conserved in prokaryotic streptomycete species as well. A vivid contrast in THN melanin biosynthesis between streptomycetes and fungi is that the THN synthesized by the action of a type III PKS is used directly for condensation in the former, while the THN synthesized by the action of type I PKSs is first reduced and the resultant 1,8-dihydroxynaphthalene is then condensed in the latter.

scholarly journals Stochastic activation and bistability in a Rab GTPase regulatory network

2020 ◽  
Vol 117 (12) ◽  
pp. 6540-6549
Author(s):  
Urban Bezeljak ◽  
Hrushikesh Loya ◽  
Beata Kaczmarek ◽  
Timothy E. Saunders ◽  
Martin Loose
Keyword(s):  
Regulatory Network ◽  
Regulatory Networks ◽  
Membrane Surface ◽  
Activity Patterns ◽  
Small Gtpases ◽  
Signaling Networks ◽  
Rab Gtpase ◽  
Endomembrane System ◽  
In Vitro Reconstitution

The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.

scholarly journals HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
Maria DeLucia ◽  
Jinwoo Ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
In Vitro Reconstitution ◽  
Hiv 1

Abstract Background Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein–protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

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