Cryo-EM studies of an ABC transporter complex (NosDFYL)
EMSL Project ID
50907
Abstract
The biological nitrogen cycle describe the enzymatic interconversion of the different chemical forms of nitrogen. In nature, microorganisms make use a huge variety of enzymes in order to perform these challenging redox reactions. One important part of the nitrogen cycle is the denitrification pathway with the last step being the reduction of nitrous oxide (N2O) to dinitrogen (N2). This reaction is catalyzed by the metalloenzyme nitrous oxide reductase present in denitrifying bacteria. N2O is well known for being a potent greenhouse gas and for its strong ozone-depleting properties that give this process a high ecological relevance.Nitrous oxide reductase contains two copper clusters, both absolutely required for catalysis. In order to make this enzyme suitable for biotechnological applications a deep understanding of its maturation pathway is crucial. During our work on the gene cluster of this enzyme from different homologs we found an ATP binding cassette (ABC) transporter NosDFY(L) highly conserved in all investigated systems. ABC transporters are a large family of integral membrane proteins that use the power of ATP hydrolysis to translocate substrates across membranes. They are present in all kingdoms of life and fulfill different roles ranging from the uptake of nutrients in bacteria to the export of drugs in human cancer cells. In our case, knock-out strains produced for Pseudomonas stutzeri revealed that the three proteins NosDFY are essential for the maturation of nitrous oxide reductase. Subsequently, we overexpressed nosDFY heterologously in E. coli and were able to isolate a stable complex NosDF2Y2. Initial cryo EM analysis of NosDFY showed a general ABC transporter architecture, consisting of two copies of the ATP binding domain (NosF) and two copies of the transmembrane domain (NosY). The additional periplasmic protein NosD is tightly bound to NosY and pointing away from the transporter suggesting to form a substrate channel. When additionally expressing nosL, an outer membrane anchored Cu chaperone, we were able to gain an even bigger complex that shows to be homogeneous when analyzed by gel filtration.
Our findings strongly suggest that the interaction of these four proteins is required for maturation of N2OR. By structural and functional investigations we hope to elucidate how the complex arrangement of NosDFYL is able to build up the copper centers of N2OR. Additionally, the structure of such a membrane spanning complex will give new insights into the architecture and function of ABC transporters in general. Different states of ATP or ATP analog as well as apo structures at high resolution will contribute to the fundamental understanding of this important superfamily. To our knowledge, this is the first isolation of an ABC transporter that is able to connect the periplasmic space in gram-negative bacteria. Looking at the membrane environment of denitrifying enzymes, this complex might even just be a small part of huge and dynamic denitrification network.
Project Details
Start Date
2019-05-15
End Date
2020-02-29
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members