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A highly conserved second-sphere active site αSer residue in nitrile hydratase (NHase), that forms a hydrogen bond with the axial metal-bound water molecule, was mutated to Ala, Asp, and Thr, in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and to Ala and Thr in the Fe-type NHase from Rhodococcus equi TG328–2 (ReNHase). All five mutants were successfully purified; metal analysis via ICP-AES indicated that all three Co-type PtNHase mutants were in their apo-form while the Fe-type αSer117Ala and αSer117Thr mutants contained 85 and 50 % of their active site Fe(III) ions, respectively. The kcat values obtained for the PtNHase mutant enzymes were between 0.03 ± 0.01 and 0.2 ± 0.02 s− 1 amounting to <0.8 % of the kcat value observed for WT PtNHase. The Fe-type ReNHase mutants retained some detectable activity with kcat values of 93 ± 3 and 40 ± 2 s− 1 for the αSer117Ala and αSer117Thr mutants, respectively, which is ~5 % of WT ReNHase activity towards acrylonitrile. UV–Vis spectra coupled with EPR data obtained on the ReNHase mutant enzymes showed subtle changes in the electronic environment around the active site Fe(III) ions, consistent with altering the hydrogen bonding interaction with the axial water ligand. X-ray crystal structures of the three PtNHase mutant enzymes confirmed the mutation and the lack of active site metal, while also providing insight into the active site hydrogen bonding network. Taken together, these data confirm that the conserved active site αSer residue plays an important catalytic role but is not essential for catalysis. They also confirm the necessity of the conserved second-sphere αSer residue for the metalation process and subsequent post-translational modification of the α-subunit in Co-type NHases but not Fe-type NHases, suggesting different mechanisms for the two types of NHases. 

Two conserved second-sphere βArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328–2 (ReNHase). Only five of the eight mutants (PtNHase βR52A, βR52K, βR157A, βR157K and ReNHase βR61A) were successfully expressed and purified. Apart from the PtNHase βR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase βR mutant enzymes were between 1.8 and 12.4 s− 1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ~10 to ~40%. UV–Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase βR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calcu- lations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second- sphere βR residues in the proposed subunit swapping process and post-translational modification of the α-sub- unit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form. 

Synopsis The shift of funding organizations to prioritize interdisciplinary work points to the need for workflow models that better accommodate interdisciplinary studies. Most scientists are trained in a specific field and are often unaware of the kind of insights that other disciplines could contribute to solving various problems. In this paper, we present a perspective on how we developed an experimental pipeline between a microscopy and image analysis/bioengineering lab. Specifically, we connected microscopy observations about a putative mechanosensing protein, obscurin, to image analysis techniques that quantify cell changes. While the individual methods used are well established (fluorescence microscopy; ImageJ WEKA and mTrack2 programs; MATLAB), there are no existing best practices for how to integrate these techniques into a cohesive, interdisciplinary narrative. Here, we describe a broadly applicable workflow of how microscopists can more easily quantify cell properties (e.g., perimeter, velocity) from microscopy videos of eukaryotic (MDCK) adherent cells. Additionally, we give examples of how these foundational measurements can create more complex, customizable cell mechanics tools and models. 

Abstract The N‐terminal half of the giant cytoskeletal protein obscurin is comprised of more than 50 Ig‐like domains, arranged in tandem. Domains 18–51 are connected to each other through short 5‐residue linkers, and this arrangement has been previously shown to form a semi‐flexible rod in solution. Domains 1–18 generally have slightly longer ~7 residue interdomain linkers, and the multidomain structure and motion conferred by this kind of linker is understudied. Here, we use NMR, SAXS, and MD to show that these longer linkers are associated with significantly more domain/domain flexibility, with the resulting multidomain structure being moderately compact. Further examination of the relationship between interdomain flexibility and linker length shows there is a 5 residue “sweet spot” linker length that results in dual‐domain systems being extended, and conversely that both longer or shorter linkers result in a less extended structure. This detailed knowledge of the obscurin N terminus structure and flexibility allowed for mathematical modeling of domains 1–18, which suggests that this region likely forms tangles if left alone in solution. Given how infrequently protein tangles occur in nature, and given the pathological outcomes that occur when tangles do arise, our data suggest that obscurin is likely either significantly scaffolded or else externally extended in the cell.