CryoEM structural exploration of catalytically active enzyme pyruvate carboxylase

DOI: 10.1038/s41467-022-33987-2

Authors: Jorge Pedro Lopez-Alonso (CIC bioGUNE, Basque Research & Technology Alliance (BRTA)) , Melisa Lazaro (CIC bioGUNE, Basque Research & Technology Alliance (BRTA)) , David Gil-Carton (CIC bioGUNE, Basque Research & Technology Alliance (BRTA); IKERBASQUE, Basque Foundation for Science) , Philip H. Choi (Columbia University) , Liang Tong (Columbia University) , Mikel Valle (CIC bioGUNE, Basque Research & Technology Alliance (BRTA))
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Communications , VOL 13

State: Published (Approved)
Published: October 2022
Diamond Proposal Number(s): 15997 , 22006

Abstract: Pyruvate carboxylase (PC) is a tetrameric enzyme that contains two active sites per subunit that catalyze two consecutive reactions. A mobile domain with an attached prosthetic biotin links both reactions, an initial biotin carboxylation and the subsequent carboxyl transfer to pyruvate substrate to produce oxaloacetate. Reaction sites are at long distance, and there are several co-factors that play as allosteric regulators. Here, using cryoEM we explore the structure of active PC tetramers focusing on active sites and on the conformational space of the oligomers. The results capture the mobile domain at both active sites and expose catalytic steps of both reactions at high resolution, allowing the identification of substrates and products. The analysis of catalytically active PC tetramers reveals the role of certain motions during enzyme functioning, and the structural changes in the presence of additional cofactors expose the mechanism for allosteric regulation.

Journal Keywords: Cryoelectron microscopy; Enzyme mechanisms; Transferases

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC)
Instruments: Krios II-Titan Krios II at Diamond , Krios III-Titan Krios III at Diamond

Added On: 24/10/2022 10:21

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Microscopy Electron Microscopy (EM) Cryo Electron Microscopy (Cryo EM)