MyJournals Home  

RSS FeedsA whole-cell, high-throughput hydrogenase assay to identify factors that modulate [NiFe]-hydrogenase activity [Microbiology] (Journal of Biological Chemistry)

 
 

18 october 2019 11:00:05

 
A whole-cell, high-throughput hydrogenase assay to identify factors that modulate [NiFe]-hydrogenase activity [Microbiology] (Journal of Biological Chemistry)
 


[NiFe]-hydrogenases have attracted attention as potential therapeutic targets or components of a hydrogen-based economy. [NiFe]-hydrogenase production is a complicated process that requires many associated accessory proteins that supply the requisite cofactors and substrates. Current methods for measuring hydrogenase activity have low throughput and often require specialized conditions and reagents. In this work, we developed a whole-cell high-throughput hydrogenase assay based on the colorimetric reduction of benzyl viologen to explore the biological networks of these enzymes in Escherichia coli. We utilized this assay to screen the Keio collection, a set of nonlethal single-gene knockouts in E. coli BW25113. The results of this screen highlighted the assay`s specificity and revealed known components of the intricate network of systems that underwrite [NiFe]-hydrogenase activity, including nickel homeostasis and formate dehydrogenase activities as well as molybdopterin and selenocysteine biosynthetic pathways. The screen also helped identify several new genetic components that modulate hydrogenase activity. We examined one E. coli strain with undetectable hydrogenase activity in more detail (?eutK), finding that nickel delivery to the enzyme active site was completely abrogated, and tracked this effect to an ancillary and unannotated lack of the fumarate and nitrate reduction (FNR) anaerobic regulatory protein. Collectively, these results demonstrate that the whole-cell assay developed here can be used to uncover new information about bacterial [NiFe]-hydrogenase production and to probe the cellular components of microbial nickel homeostasis.


 
213 viewsCategory: Biochemistry
 
Muscle metabolic reprogramming underlies the resistance of liver fatty acid-binding protein (LFABP)-null mice to high-fat feeding-induced decline in exercise capacity [Lipids] (Journal of Biological Chemistry)
The mammalian host protein DAP5 facilitates the initial round of translation of Coxsackievirus B3 RNA [Protein Synthesis and Degradation] (Journal of Biological Chemistry)
 
 
blog comments powered by Disqus


MyJournals.org
The latest issues of all your favorite science journals on one page

Username:
Password:

Register | Retrieve

Search:

Biochemistry


Copyright © 2008 - 2024 Indigonet Services B.V.. Contact: Tim Hulsen. Read here our privacy notice.
Other websites of Indigonet Services B.V.: Nieuws Vacatures News Tweets Nachrichten