8FOL

The structure of a crystallizable variant of E. coli pyruvate formate-lyase activating enzyme bound to SAM, alternate crystal form

Summary for 8FOL

• Entry DOI: 10.2210/pdb8fol/pdb
• Related: 8FO0
• Descriptor: Pyruvate formate-lyase 1-activating enzyme, IRON/SULFUR CLUSTER, S-ADENOSYLMETHIONINE, ... (6 entities in total)
• Functional Keywords: radical sam, activase, pfl, oxidoreductase
• Biological source: Escherichia coli K-12
• Total number of polymer chains: 1
• Total formula weight: 29132.81

Authors

Moody, J.D.,Saxton, A.J.,Galambas, A.,Lawrence, C.M.,Broderick, J.B. (deposition date: 2022-12-31, release date: 2023-05-24, Last modification date: 2024-05-22)

Primary citation

Moody, J.D.,Hill, S.,Lundahl, M.N.,Saxton, A.J.,Galambas, A.,Broderick, W.E.,Lawrence, C.M.,Broderick, J.B.
Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage.
J.Biol.Chem., 299:104791-104791, 2023

PubMed Abstract: Radical S-adenosyl-l-methionine (SAM) enzymes are ubiquitous in nature and carry out a broad variety of difficult chemical transformations initiated by hydrogen atom abstraction. Although numerous radical SAM (RS) enzymes have been structurally characterized, many prove recalcitrant to crystallization needed for atomic-level structure determination using X-ray crystallography, and even those that have been crystallized for an initial study can be difficult to recrystallize for further structural work. We present here a method for computationally engineering previously observed crystallographic contacts and employ it to obtain more reproducible crystallization of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE). We show that the computationally engineered variant binds a typical RS [4Fe-4S] cluster that binds SAM, with electron paramagnetic resonance properties indistinguishable from the native PFL-AE. The variant also retains the typical PFL-AE catalytic activity, as evidenced by the characteristic glycyl radical electron paramagnetic resonance signal observed upon incubation of the PFL-AE variant with reducing agent, SAM, and PFL. The PFL-AE variant was also crystallized in the [4Fe-4S] state with SAM bound, providing a new high-resolution structure of the SAM complex in the absence of substrate. Finally, by incubating such a crystal in a solution of sodium dithionite, the reductive cleavage of SAM is triggered, providing us with a structure in which the SAM cleavage products 5'-deoxyadenosine and methionine are bound in the active site. We propose that the methods described herein may be useful in the structural characterization of other difficult-to-resolve proteins.

PubMed: 37156396
DOI: 10.1016/j.jbc.2023.104791

Experimental method

X-RAY DIFFRACTION (2.65 Å)