8fsi
From Proteopedia
The structure of a crystallizable variant of E. coli pyruvate formate-lyase activating enzyme bound to SAM
Structural highlights
FunctionPFLA_ECOLI Activation of pyruvate formate-lyase 1 under anaerobic conditions by generation of an organic free radical, using S-adenosylmethionine and reduced flavodoxin as cosubstrates to produce 5'-deoxy-adenosine. Publication Abstract from PubMedRadical 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](2+/+) cluster that binds SAM, with electron paramagnetic resonance (EPR) properties indistinguishable from the native PFL-AE. The variant also retains the typical PFL-AE catalytic activity, as evidenced by the characteristic glycyl radical EPR 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](2+) 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. Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage.,Moody JD, Hill S, Lundahl MN, Saxton AJ, Galambas A, Broderick WE, Lawrence CM, Broderick JB J Biol Chem. 2023 May 6:104791. doi: 10.1016/j.jbc.2023.104791. PMID:37156396[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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