An official website of the United States government
Human ornithine transcarbamylase (hOTC) is a mitochondrial transferase protein involved in the urea cycle and is crucial for the conversion of toxic ammonia to urea. Structural analysis coupled with kinetics studies of E. coli, rat, bovine and other transferase proteins have identified residues that play key roles in substrate recognition and conformational changes but do not provide direct evidence for all the active residues involved in OTC function. Here, computational methods were used to predict likely active residues of hOTC; the function of these residues was then probed with site-directed mutagenesis and biochemical characterization. This process identified previously reported active residues as well as distal residues that contribute to activity. Mutation of active site residue D263 resulted in substantial loss of activity without a decrease in protein stability, suggesting a key catalytic role for this residue. Mutation of predicted second-layer residues H302, K307, and E310 resulted in significant decreases in enzymatic activity relative to wild-type (WT) hOTC with respect to L-ornithine. Mutation of fourth-layer residue H107 to the hOTC H107N variant resulted in a 66-fold decrease in catalytic efficiency relative to WT hOTC with respect to carbamoyl phosphate, and a substantial loss of thermal stability. Further investigation identified H107 and to a lesser extent E98Q as key residues involved in maintaining hOTC quaternary structure. This work demonstrates biochemically the importance of D263 in hOTC catalytic activity and shows that residues remote from the active site also play key roles in activity.
more »
« less
Distal residues contribute to enzymatic catalysis in human phosphoglucose isomerase through modulation of dynamics and electrostatics
Distal residues in enzymes, although spatially remote from the active site, play critical roles in modulating catalytic efficiency through structural dynamics and electrostatics. In this study, we investigate the contribution of distal residues to the catalytic activity of human phosphoglucose isomerase (hPGI) using molecular dynamics simulations and electrostatic calculations. Six key residues located in the second and third layers around the active site were studied previously through systematic mutagenesis. Variants, including those in the second shell K362A, Q388A, E495Q, and D511N and those in the third shell H100L and H396L, were previously reported to show significant reductions in catalytic turnover and efficiency. Analysis of structural dynamics demonstrates increased flexibility in helices critical for maintaining active site geometry, including helix-17 and helix-18, leading to destabilization of catalytic residues E358, R273, and H389′. In addition, the important interaction between H389′ and E217, essential for the ring-opening step, was disrupted across all variants. Electrostatic perturbations, including altered protonation equilibria and pKa values of catalytic residues, further impede key steps of the catalytic mechanism, such as ligand binding, ring opening, and isomerization. These findings reveal the complex interplay between distal residues, structural dynamics, and electrostatics in regulating hPGI’s catalytic mechanism, providing insights into enzyme function and guiding future enzyme engineering and drug design efforts.
more »
« less
- Award ID(s):
- 2147498
- PAR ID:
- 10702032
- Publisher / Repository:
- AIP
- Date Published:
- Journal Name:
- The Journal of Chemical Physics
- Volume:
- 163
- Issue:
- 1
- ISSN:
- 0021-9606
- Subject(s) / Keyword(s):
- phosphoglucose isomerase distal residues conformational effects enzyme electrostatics accelerated molecular dynamics Partial Order Optimum Likelihood
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5–5, RA95.5–8, and RA95.5–8F, representing progress along an evolutionary trajectory with increasing activity, is examined. Computed measures of coupling between charged states of residues show that, as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues is increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, are tested using site-directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants show decreases in kcat relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibit significant decreases in kcat/KM across the series. Only modest decreases in kcat/KM are observed for the E85Q variants with little effect on kcat. Computed metrics of the degree of coupling between protonation states rise significantly as evolution proceeds and catalytic turnover rate increases. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst.more » « less
-
The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.more » « less
-
The kynurenine pathway is the primary route for L-tryptophan degradation in mammals. Intermediates and side products of this pathway are involved in immune response and neurodegenerative diseases. This makes the study of enzymes, especially those from mammalian sources, of the kynurenine pathway worthwhile. Recent studies on a bacterial version of an enzyme of this pathway, 2-aminomuconate semialdehyde (2-AMS) dehydrogenase (AMSDH), have provided a detailed understanding of the catalytic mechanism and identified residues conserved for muconate semialdehyde recognition and activation. Findings from the bacterial enzyme have prompted the reconsideration of the function of a previously identified human aldehyde dehydrogenase, ALDH8A1 (or ALDH12), which was annotated as a retinal dehydrogenase based on its ability to preferentially oxidize 9-cis-retinal over trans-retinal. Here, we provide compelling bioinformatics and experimental evidence that human ALDH8A1 should be reassigned to the missing 2-AMS dehydrogenase of the kynurenine metabolic pathway. For the first time, the product of the semialdehyde oxidation by AMSDH is also revealed by NMR and high-resolution MS. We found that ALDH8A1 catalyzes the NAD+ -dependent oxidation of 2- AMS with a catalytic efficiency equivalent to that of AMSDH from the bacterium Pseudomonas fluorescens. Substitution of active-site residues required for substrate recognition, binding, and isomerization in the bacterial enzyme resulted in human ALDH8A1 variants with 160-fold increased Km or no detectable activity. In conclusion, this molecular study establishes an additional enzymatic step in an important human pathway for tryptophan catabolism.more » « less
-
Abstract Phosphodiesterase‐5 (PDE5) is responsible for regulating the concentration of the second messenger molecule cGMP by hydrolyzing it into 5′‐GMP. PDE5 is implicated in erectile dysfunction and cardiovascular diseases. The substrate binding site in the catalytic domain of PDE5 is surrounded by several dynamic structural motifs (including the α14 helix, M‐loop, and H‐loop) that are known to switch between inactive and active conformational states via currently unresolved structural intermediates. We evaluated the conformational dynamics of these structural motifs in the apo state and upon binding of an allosteric inhibitor ( evodiamine ) or avanafil , a competitive inhibitor. We employed enhanced sampling‐based replica exchange solute scaling (REST2) method, principal component analysis (PCA), time‐lagged independent component analysis (tICA), molecular dynamics (MD) simulations, and well‐tempered metadynamics simulations to probe the conformational changes in these structural motifs. Our results support a regulatory mechanism for PDE5, where the α14 helix alternates between an inward (lower activity) conformation and an outward (higher activity) conformation that is accompanied by the folding/unfolding of the α8′ and α8″ helices of the H‐loop. When the allosteric inhibitor evodiamine is bound to PDE5, the inward (inactive) state of the α14 helix is preferred, thus preventing substrate access to the catalytic site. In contrast, competitive inhibitors of PDE5 block catalysis by occupying the active site accompanied by stabilization of the outward conformation of the α14 helix. Defining the conformational dynamics underlying regulation of PDE5 activation will be helpful in rational design of next‐generation small molecules modulators of PDE5 activity.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1063/5.0274141
