Dataset Information

Enhancing Mn(II)-Binding and Manganese Peroxidase Activity in a Designed Cytochrome c Peroxidase through Fine-Tuning Secondary-Sphere Interactions.

ABSTRACT: Noncovalent second-shell interactions are important in controlling metal-binding affinity and activity in metalloenzymes, but fine-tuning these interactions in designed metalloenzymes has not been fully explored. As a result, most designed metalloenzymes have low metal-binding affinity and activity. Here we identified three mutations in the second coordination shell of an engineered Mn(II)-binding site in cytochrome c peroxidase (called MnCcP.1, containing Glu45, Glu37, and Glu181 ligands) that mimics the native manganese peroxidase (MnP), and explored their effects on both Mn(II)-binding affinity and MnP activity. First, removing a hydrogen bond to Glu45 through Tyr36Phe mutation enhanced Mn(II)-binding affinity, as evidenced by a 2.8-fold decrease in the KM of Mn(II) oxidation. Second, introducing a salt bridge through Lys179Arg mutation improved Glu35 and Glu181 coordination to Mn(II), decreasing KM 2.6-fold. Third, eliminating a steric clash that prevented Glu37 from orienting toward Mn(II) resulted in an 8.6-fold increase in kcat/KM, arising primarily from a 3.6-fold decrease in KM, with a KM value comparable to that of the native enzyme (0.28 mM vs 0.19 mM for Pleurotus eryngii MnP PS3). We further demonstrated that while the effects of Tyr36Phe and Lys179Arg mutations are additive, because involved in secondary-shell interactions to different ligands, other combinations of mutations were antagonistic because they act on different aspects of the Mn(II) coordination at the same residues. Finally, we showed that these MnCcP variants are functional models of MnP that mimic its activity in both Mn(II) oxidation and degradation of a phenolic lignin model compound and kraft lignin. In addition to achieving KM in a designed protein that is similar to the that of native enzyme, our results offer molecular insight into the role of noncovalent interactions around metal-binding sites for improving metal binding and overall activity; such insight can be applied to rationally enhance these properties in other metalloenzymes and their models.

SUBMITTER: Hosseinzadeh P

PROVIDER: S-EPMC5178831 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Enhancing Mn(II)-Binding and Manganese Peroxidase Activity in a Designed Cytochrome c Peroxidase through Fine-Tuning Secondary-Sphere Interactions.

Hosseinzadeh Parisa P Mirts Evan N EN Pfister Thomas D TD Gao Yi-Gui YG Mayne Christopher C Robinson Howard H Tajkhorshid Emad E Lu Yi Y

Biochemistry 20160302 10

Noncovalent second-shell interactions are important in controlling metal-binding affinity and activity in metalloenzymes, but fine-tuning these interactions in designed metalloenzymes has not been fully explored. As a result, most designed metalloenzymes have low metal-binding affinity and activity. Here we identified three mutations in the second coordination shell of an engineered Mn(II)-binding site in cytochrome c peroxidase (called MnCcP.1, containing Glu45, Glu37, and Glu181 ligands) that ...[more]

PMID: 26885726

Similar Datasets

Project description:BackgroundDecoding human genomic sequences requires comprehensive analysis of DNA sequence functionality. Through computational and experimental approaches, researchers have studied the genotype-phenotype relationship and generate important datasets that help unravel complicated genetic blueprints. Thus, the recently developed artificial intelligence methods can be used to interpret the functions of those DNA sequences.MethodsThis study explores the use of deep learning, particularly pre-trained genomic models like DNA_bert_6 and human_gpt2-v1, in interpreting and representing human genome sequences. Initially, we meticulously constructed multiple datasets linking genotypes and phenotypes to fine-tune those models for precise DNA sequence classification. Additionally, we evaluate the influence of sequence length on classification results and analyze the impact of feature extraction in the hidden layers of our model using the HERV dataset. To enhance our understanding of phenotype-specific patterns recognized by the model, we perform enrichment, pathogenicity and conservation analyzes of specific motifs in the human endogenous retrovirus (HERV) sequence with high average local representation weight (ALRW) scores.ResultsWe have constructed multiple genotype-phenotype datasets displaying commendable classification performance in comparison with random genomic sequences, particularly in the HERV dataset, which achieved binary and multi-classification accuracies and F1 values exceeding 0.935 and 0.888, respectively. Notably, the fine-tuning of the HERV dataset not only improved our ability to identify and distinguish diverse information types within DNA sequences but also successfully identified specific motifs associated with neurological disorders and cancers in regions with high ALRW scores. Subsequent analysis of these motifs shed light on the adaptive responses of species to environmental pressures and their co-evolution with pathogens.ConclusionsThese findings highlight the potential of pre-trained genomic models in learning DNA sequence representations, particularly when utilizing the HERV dataset, and provide valuable insights for future research endeavors. This study represents an innovative strategy that combines pre-trained genomic model representations with classical methods for analyzing the functionality of genome sequences, thereby promoting cross-fertilization between genomics and artificial intelligence.

Project description:N-substituted glycine oligomers, or peptoids, have emerged as an important class of foldamers for the study of biomolecular interactions and for potential use as therapeutic agents. However, the design of peptoids with well-defined conformations a priori remains a formidable challenge. New approaches are required to address this problem, and the systematic study of the role of individual monomer units in the global peptoid folding process represents one strategy. Here, we report our efforts toward this approach through the design, synthesis, and characterization of peptoids containing nitroaromatic monomer units. This work required the synthesis of a new chiral amine building block, (S)-1-(2-nitrophenyl)ethanamine (s2ne), which could be readily installed into peptoids using standard solid-phase peptoid synthesis techniques. We designed a series of peptoid nonamers that allowed us to probe the effects of this relatively electron-deficient and sterically encumbered alpha-chiral side chain on peptoid structure, namely, the peptoid threaded loop and helix. Circular dichroism spectroscopy of the peptoids revealed that the nitroaromatic monomer has a significant effect on peptoid secondary structure. Specifically, the threaded loop structure was disrupted in a nonamer containing alternating N-(S)-1-phenylethylglycine (Nspe) and Ns2ne monomers, and the major conformation was helical instead. Indeed, placement of a single Ns2ne at the N-terminal position of (Nspe)(9) resulted in a destabilized form of the threaded loop structure relative to the homononamer (Nspe)(9). Conversely, we observed that incorporation of N-(S)-1-(4-nitrophenyl)ethylglycine (Nsnp, a p-nitro monomer) at the N-terminal position stabilized the threaded loop structure relative to (Nspe)(9). Additional experiments revealed that nitroaromatic side chains can influence peptoid nonamer folding by modulating the strength of key intramolecular hydrogen bonds in the peptoid threaded loop structure. Steric interactions were also implicated for the Ns2ne monomer. Overall, this study provides further evidence that aromatic side-chain structure, even if perturbed in a single monomer unit, can strongly influence local peptoid backbone conformation.

Dataset Information

Enhancing Mn(II)-Binding and Manganese Peroxidase Activity in a Designed Cytochrome c Peroxidase through Fine-Tuning Secondary-Sphere Interactions.

Publications

Enhancing Mn(II)-Binding and Manganese Peroxidase Activity in a Designed Cytochrome c Peroxidase through Fine-Tuning Secondary-Sphere Interactions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets