An official website of the United States government
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.
more »
« less
This content will become publicly available on January 1, 2026
Catalytic and post-translational maturation roles of a conserved active site serine residue in nitrile hydratases
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.
more »
« less
- PAR ID:
- 10552590
- Editor(s):
- NA
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Journal of Inorganic Biochemistry
- Volume:
- 262
- Issue:
- C
- ISSN:
- 0162-0134
- Page Range / eLocation ID:
- 112763
- Subject(s) / Keyword(s):
- Nitrile hydratase Cobalt Metallochaperone Mutagenesis Serine
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
The strictly conserved αSer162 residue in the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSerA) that exhibited no observable activity, and the second (αSerB) contained its full complement of cobalt ions and was active with a kcat value of 63 ± 3 s−1 towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSerA was determined at 1.85 Å resolution and contained no detectable cobalt per α2β2 heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type PtNHase, but were able to hydrate acrylonitrile with kcat values of 120 ± 6, 29 ± 3, and 14 ± 1 s−1 for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases.more » « less
-
null (Ed.)To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = −260 mV) and mutant Mb (E° = −300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.more » « less
-
Abstract With the aim of constructing hydrogen‐bonding networks in synthetic complexes, two new ligands derived fromcis,cis‐1,3,5‐triaminocyclohexane (TACH) have been prepared that feature pendant pyrrole or indole rings as outer‐sphere H‐bond donors. The TACH framework offers a facial arrangement of threeN‐donors, thereby mimicking common coordination motifs in the active sites of nonheme Fe and Cu enzymes. X‐ray structural characterization of a series of CuI‐X complexes (X=F, Cl, Br, NCS) revealed that these neutral ligands (H3LR, R=pyrrole or indole) coordinate in the intended facialN3manner, yielding four‐coordinate complexes with idealizedC3symmetry. The N−H units of the outer‐sphere heterocycles form a hydrogen‐bonding cavity around the axial (pseudo)halide ligand, as verified by crystallographic, spectroscopic, and computational analyses. Treatment of H3Lpyrroleand H3Lindolewith divalent transition metal chlorides (MIICl2, M=Fe, Cu, Zn) causes one heterocycle to deprotonate and coordinate to the M(II) center, giving rise to tetradentate ligands with two remaining outer‐sphere H‐bond donors. Further ligand deprotonation is observed upon reaction with Ni(II) and Cu(II) salts with weakly coordinating counteranions. The reported complexes highlight the versatility of TACH‐based ligands with pendant H‐bond donors, as the resulting scaffolds can support multiple protonation states, coordination geometries, and H‐bonding interactions.more » « less
-
Abstract Hydrogen bonds (H‐bonds) have been shown to modulate the chemical reactivities of iron centers in iron‐containing dioxygen‐activating enzymes and model complexes. However, few examples are available that investigate how systematic changes in intramolecular H‐bonds within the secondary coordination sphere influence specific properties of iron intermediates, such as iron‐oxido/hydroxido species. Here, we used57Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the Fe‐O/OH vibrations in a series of FeIII‐hydroxido and FeIV/III‐oxido complexes with varying H‐bonding networks but having similar trigonal bipyramidal primary coordination spheres. The data show that even subtle changes in the H‐bonds to the Fe‐O/OH units result in significant changes in their vibrational frequencies, thus demonstrating the utility of NRVS in studying the effect of the secondary coordination sphere to the reactivities of iron complexes.more » « less
