The thioesterase of the erythromycin-producing polyketide synthase: mechanistic studies in vitro to investigate its mode of action and substrate specificity

1995 ◽  
pp. 1519 ◽  
Author(s):  
Ranjana Aggarwal ◽  
Patrick Caffrey ◽  
Peter F. Leadlay ◽  
Cameron J. Smith ◽  
James Staunton
Keyword(s):  
Substrate Specificity ◽  
Mode Of Action ◽  
Polyketide Synthase ◽  
Mechanistic Studies

scholarly journals An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors

2014 ◽  
Vol 95 (12) ◽  
pp. 2683-2692 ◽  
Author(s):  
Irina C. Albulescu ◽  
Ali Tas ◽  
Florine E. M. Scholte ◽  
Eric J. Snijder ◽  
Martijn J. van Hemert
Keyword(s):  
Mode Of Action ◽  
Chikungunya Virus ◽  
Rna Synthesis ◽  
Mechanistic Studies ◽  
Vitro Assay ◽  
Virus Rna ◽  
Infected Cells ◽  
Transcription Complexes ◽  
Crude Membrane Fraction

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay 32P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5′ end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15 000 . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3′dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

scholarly journals Broadening substrate specificity of a chain-extending ketosynthase through a single active-site mutation

2016 ◽  
Vol 52 (54) ◽  
pp. 8373-8376 ◽  
Author(s):  
Annabel C. Murphy ◽  
Hui Hong ◽  
Steve Vance ◽  
R. William Broadhurst ◽  
Peter F. Leadlay
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Polyketide Synthase ◽  
Model System ◽  
Site Mutation ◽  
Ketosynthase Domain ◽  
Substrate Tolerance ◽  
A Chain

An in vitro model system based on a ketosynthase domain of the erythromycin polyketide synthase was used to probe the apparent substrate tolerance of ketosynthase domains of the mycolactone polyketide synthase.

scholarly journals The Mode of Action of Dibutyryl Adenosine 3′,5′-Monophosphate on Bone Tissue in Vitro

1971 ◽  
Vol 246 (22) ◽  
pp. 6770-6775
Author(s):  
Johannes N.M. Heersche ◽  
Susan A. Fedak ◽  
G.D. Aurbach
Keyword(s):  
Bone Tissue ◽  
Mode Of Action

scholarly journals Characterization of Phospholipase Activity of the Pseudomonas aeruginosa Type III Cytotoxin, ExoU

2005 ◽  
Vol 187 (3) ◽  
pp. 1192-1195 ◽  
Author(s):  
Hiromi Sato ◽  
Jimmy B. Feix ◽  
Cecilia J. Hillard ◽  
Dara W. Frank
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Yeast Extract ◽  
Enzyme Activation ◽  
Phospholipase Activity ◽  
Eukaryotic Protein ◽  
Type Iii

ABSTRACT Recombinant ExoU (rExoU) and yeast extract were used to optimize an in vitro phospholipase assay as a basis for identifying the mechanism for enzyme activation and substrate specificity. Our results support a model in which a eukaryotic protein cofactor or complex facilitates the interaction of rExoU with phospholipid substrates.

scholarly journals The in vivo and in vitro substrate specificity of quinoprotein glucose dehydrogenase ofAcinetobacter calcoaceticusLMD79.41

1987 ◽  
Vol 43 (2) ◽  
pp. 195-200 ◽  
Author(s):  
P. Dokter ◽  
J.T. Pronk ◽  
B.J. Schie ◽  
J.P. Dijken ◽  
J.A. Duine
Keyword(s):  
Substrate Specificity ◽  
Glucose Dehydrogenase
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