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.
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A Case Study of the Glycoside Hydrolase Enzyme Mechanism Using an Automated QM-Cluster Model Building Toolkit
Glycoside hydrolase enzymes are important for hydrolyzing the β-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism of Glycoside Hydrolase Family 7 (GH7) was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol −1 for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction; and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol −1 for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme.
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- Award ID(s):
- 1846408
- NSF-PAR ID:
- 10337984
- Date Published:
- Journal Name:
- Frontiers in Chemistry
- Volume:
- 10
- ISSN:
- 2296-2646
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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This work probed the thermal “switchability” from ethylene coordination/insertion to controlled radical polymerization of methyl acrylate (MA) for Brookhart-type α-diimine PdII catalysts. The investigation focused on the extremely bulky 2,6-bis(3,5-dimethylphenyl)-4-methylphenyl (Xyl4Ph) α-diimine N-substituents to probe reversible PdII–C bond activation in the MA-quenched Pd-capped PE intermediate and reversible trapping during radical MA polymerization. The substituent steric effect on the relative stability of various [PE–MA–PdII(ArN═CMeCMe═NAr)]+ chain-end structures and on the bond dissociation-free energy (BDFE) for the homolytic PdII–C bond cleavage has been assessed by DFT calculations at the full quantum mechanics (QM) and QM/molecular mechanics (QM/MM) methods. The structures comprise ester-chelated forms with the Pd atom bonded to the α, β, and γ C atoms as a result of 2,1 MA insertion into the PE–Pd bond and of subsequent chain walking, as well as related monodentate (ring-opened) forms resulting from the addition of MA or acetonitrile. The opened Cα-bonded form is electronically favored for smaller N-substituents, including 2,6-diisopropylphenyl (Dipp), particularly when MeCN is added, but the open Cγ-bonded form is preferred for the extremely bulky system with Ar = Xyl4Ph. The Pdα–C bond is the weakest one to cleave, with the BDFE decreasing as the Ar steric bulk is increased (31.8, 25.8, and 12.6 kcal mol–1 for Ph, Dipp, and Xyl4Ph, respectively). However, experimental investigations on the [PE–MA–PdII(ArN═CMeCMe═NAr)]+ (Ar = Xyl4Ph) macroinitiator do not show any evidence of radical formation under thermal activation conditions, while photolytic activation produces both TEMPO-trapped (TEMPO = 2,2,6,6-tetramethylpiperidinyloxy) and unsaturated MA-containing PE chains. The DFT investigation has highlighted a low-energy pathway for termination of the PE–MA• radicals by disproportionation, promoted by β-H elimination/dissociation and H-atom abstraction from the PdII–H intermediate by a second radical. This phenomenon appears to be the main reason for the failure of this PdII system to control the radical polymerization of MA by the OMRP (OMRP = organometallic-mediated radical polymerization) mechanism.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fchem.2022.854318
