Biology subjects

Follmer, A. H.

Publications and source records attributed to Follmer, A. H..

4 recordsLinked to original sources

bioRxiv2026

Structural Determinants of Catalytic Directionality in an AMP-Forming Acetyl-CoA Synthetase from Syntrophus aciditrophicus

Acetyl-coenzyme A (CoA) is a central metabolic intermediate that links carbon and energy metabolism across all domains of life. The interconversion of acetate and acetyl-CoA is carried out by three enzyme pathways: acetate kinase/phosphotransacetylase, ADP-forming acetyl-CoA synthetase, and AMP-forming acetyl-CoA synthetase (Acs). Acs enzymes serve critical physiological roles across diverse organisms by catalyzing a reversible two-step reaction forming acetyl-CoA and AMP from acetate and ATP. Isolated from the wastewater reclamation facility in Norman, Oklahoma, Syntrophus aciditrophicus strain SB (Sa) thermodynamically favors synthesizing acetate and ATP from acetyl-CoA and AMP using an AMP-forming acetyl-CoA synthetase (SaAcs1). The origin of the preference for AMP formation and the structural determinants of both the thioester-forming step and catalytic bias remain poorly understood. Here, we report a 2.2 [A] crystal structure of full-length SaAcs1 in the adenylation conformation with acetyl-AMP bound in the active site. Structural comparison to the extensively characterized Acs enzymes from Salmonella enterica (SeAcs) and Cryptococcus neoformans (CnAcs) revealed a displaced CoA-binding loop in SaAcs1. Enzymatic assays support that SaAcs1 preferentially catalyzes the ATP-forming reaction. Site-directed mutagenesis demonstrated that reversion of two residues, G196 and T197, at the beginning of the CoA-binding loop to the consensus sequence repositions the loop and shifts catalytic preference toward the AMP-forming direction. Together, these results establish the CoA-binding loop and G196 and T197 as the primary structural determinants of catalytic bias in SaAcs1.

biochemistry↗

bioRxiv2026

Conformational flexibility of soybean lipoxygenase is coupled to crystal solvent content in serial crystallography

X-ray crystallography is increasingly employed to study protein conformational ensembles under physiological conditions, but the effects of the crystal lattice on protein motions remains understudied. Here, we report the structure determination of soybean lipoxygenase-1 (SLO) from microcrystal slurries using serial femtosecond crystallography (SFX) at the Linac Coherent Light Source. During data analysis, we observed unexpected polymorphism in SLOs unit-cell parameters, arising from two compounding factors: indexing ambiguities caused by the pseudo-tetragonal symmetry of the SLO crystal lattice, and true non-isomorphism between individual crystal populations consistent with different solvent content. By combining unit-cell clustering with systematic reindexing, we resolved two distinct polymorphs and determined two independent structures from a single experiment. The two structures exhibit a small overall RMSD (0.34 [A]), yet a difference distance matrix reveals coordinated rearrangements that are not readily apparent from simple structural overlays. Furthermore, a difference of approximately 8.5% in crystal solvent content produces measurable differences in crystal contacts and conformational flexibility. The more hydrated (large-cell) polymorph exhibits greater inter-domain flexibility, as well as higher B-factors in key hydrophobic core residues. In the dehydrated (small-cell) polymorph, which shows less interdomain flexibility, these same residues adopt discrete alternative conformations resolvable in the electron density. Our results highlight that subtle changes in crystal packing can give rise to distinct conformational landscapes for crystallized proteins, with potential implications for the interpretation of protein intramolecular dynamics from crystallographic data. SynopsisUsing soybean lipoxygenase-1 as a model system, we determine two independent structures within a single experiment that differ in crystal solvent content. Despite a small overall RMSD, a detailed analysis reveals coordinated domain-level rearrangements and shows that conformational flexibility is sensitive to crystal packing, a consideration of broad relevance for the study of protein dynamics using X-ray crystallography.

biochemistry↗

bioRxiv2026

Structural Basis of a Novel Heme Binding Bacterial One-Component Switch

One-component systems (OCSs) integrate sensory and effector functions within a single protein, enabling rapid gene expression changes in response to environmental cues. Here, we characterized a novel heme binding OCS protein, FG214, from Fimbriimonas ginsengisoli, a redox-regulated helix-turn-helix transcription factor in which heme iron ligand state controls a monomer-to-dimer switch. Data supporting this included our observation of the FG214 PAS domain binding a hexacoordinate heme b in oxidized conditions and undergoing a slate of redox and ligand-dependent conformational changes, transitioning from a monomer to a homodimer. Spectroscopic and structural data revealed that oxidation stabilizes the likely HTH-PAS intramolecular domain interface, while reduction of the heme iron dissociates the HTH, freeing previously-sequestered homodimerization surfaces. Similar effects were seen by addition of a small molecule ferric heme ligand, as directly visualized with a 1.47 [A] crystal structure of an imidazole-bound truncated construct. Using in vitro DNA-binding assays, we identified an artificial promoter sequence and demonstrated ligand-enhanced protein-DNA binding. Finally, we performed in vivo proof of concept experiments establishing FG214 as a redox-sensitive scaffold for biosensor engineering. Together, these findings define FG214 as a novel heme-binding PAS DNA binding protein, complementing known heme-PAS two-component signaling switches.

biochemistry↗

bioRxiv2023

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

biochemistry↗