Visualizing One-Metal-Ion-Dependent and Histidine-Promoted DNA Hydrolysis by the His-Me Family I-PpoI Nuclease

Core Concepts

One divalent metal ion and a conserved histidine residue are sufficient to promote water deprotonation and nucleophilic attack during DNA hydrolysis by the His-Me family I-PpoI nuclease.

Abstract

The study used time-resolved crystallography to observe the catalytic process of the His-Me family I-PpoI nuclease and investigate its metal ion dependence and water deprotonation mechanism.
Key highlights:

Only one divalent metal ion (Mg2+ or Mn2+) was captured in the I-PpoI active site during DNA hydrolysis, even at high metal ion concentrations. This single metal ion is located on the leaving group side of the scissile phosphate.
The conserved histidine residue (His98) is the primary proton acceptor that deprotonates the nucleophilic water, but alternative pathways involving another histidine (His78) or surrounding waters can also activate the nucleophile in the absence of His98.
Metal ion binding and water deprotonation are highly concerted during catalysis. Perturbing the deprotonation pathway, such as by mutating His98, affects metal ion binding, suggesting a coupled mechanism.
The study provides mechanistic insights into one-metal-ion-dependent nucleases, which differ from the two-metal-ion mechanisms observed in DNA polymerases and some other nucleases.

Stats

The electron density (r.m.s.d) of the product phosphate and metal ion strongly correlated (R2 > 0.95) during the reaction process, indicating that saturation of the single metal ion site is required and sufficient for catalysis.
The concentration of Mg2+ required for 50% product formation increased over 100-fold when the pH was lowered from 8 to 6, suggesting that pH affects metal ion binding.

Quotes

"One and only one divalent metal ion was captured within the I-PpoI active site during catalysis, even when high concentration (200 mM Mn2+) of metal ion was tested."
"Perturbing the deprotonation pathway, such as by mutating His98, affects metal ion binding, suggesting a coupled mechanism."

Key Insights Distilled From

Observing one-divalent-metal-ion dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo

by Chang,C., Zh... at www.biorxiv.org 05-05-2024

https://www.biorxiv.org/content/10.1101/2024.05.02.592236v2

Deeper Inquiries

How do the active site configurations and catalytic mechanisms of one-metal-ion-dependent nucleases compare to those of two-metal-ion-dependent nucleases and polymerases

The active site configurations and catalytic mechanisms of one-metal-ion-dependent nucleases differ from those of two-metal-ion-dependent nucleases and polymerases in several key ways. One-metal-ion-dependent nucleases, such as the His-Me family nucleases, typically have a single divalent metal ion in their active site, which is coordinated by water molecules and specific amino acid residues. This metal ion plays a crucial role in stabilizing the transition state of the reaction and promoting catalysis. In contrast, two-metal-ion-dependent nucleases and polymerases have two metal ions in their active sites, each serving a specific function. One metal ion (Me2+A) stabilizes the leaving group, while the other metal ion (Me2+B) interacts with the nucleophile and helps in catalysis.
Furthermore, in one-metal-ion-dependent nucleases, the active site typically contains a conserved histidine residue that assists in deprotonating a nearby water molecule for nucleophilic attack. This histidine residue is essential for catalysis and is involved in promoting the reaction. In contrast, two-metal-ion-dependent nucleases and polymerases may have different amino acid residues involved in coordinating the metal ions and promoting catalysis. The presence of two metal ions allows for more complex coordination and stabilization of the reaction intermediates, leading to efficient catalysis.
Overall, the active site configurations and catalytic mechanisms of one-metal-ion-dependent nucleases are simpler compared to two-metal-ion-dependent nucleases and polymerases, but they are equally effective in catalyzing DNA hydrolysis through a concerted mechanism involving a single metal ion and specific amino acid residues.

What are the potential limitations or drawbacks of using a single metal ion for DNA hydrolysis, and how might nature have evolved strategies to overcome these

Using a single metal ion for DNA hydrolysis in nucleases may have potential limitations or drawbacks that could impact catalytic efficiency. One limitation is the need for precise coordination and stabilization of the metal ion in the active site to ensure optimal catalytic activity. The presence of only one metal ion means that there is less flexibility in the catalytic mechanism compared to two-metal-ion-dependent enzymes. Additionally, the single metal ion must be able to perform multiple functions, such as stabilizing the transition state, promoting nucleophilic attack, and facilitating product release, which can be challenging.
Nature has evolved strategies to overcome these limitations and ensure efficient catalysis by one-metal-ion-dependent nucleases. For example, specific amino acid residues, such as histidine, asparagine, and other coordinating residues, play crucial roles in assisting the metal ion in catalysis. These residues help in orienting the substrate, promoting deprotonation of the nucleophilic water, and stabilizing the transition state. Additionally, the active site architecture of these nucleases is highly conserved, allowing for efficient coordination of the metal ion and substrate.
Furthermore, the concerted mechanism of metal ion binding and water deprotonation observed in this study suggests that nature has optimized the catalytic process to ensure efficient DNA hydrolysis. By coordinating the binding of the metal ion with the deprotonation of the nucleophilic water, nucleases can effectively catalyze DNA cleavage with high specificity and efficiency.

Could the insights from this study on the concerted metal ion binding and water deprotonation be applied to engineer more efficient nucleases for genome editing or other biotechnological applications

The insights from this study on the concerted metal ion binding and water deprotonation in one-metal-ion-dependent nucleases could be applied to engineer more efficient nucleases for genome editing and other biotechnological applications. By understanding the specific roles of the metal ion and amino acid residues in catalysis, researchers can design novel nucleases with enhanced catalytic activity and specificity.
One potential application of this knowledge is in the development of CRISPR-Cas systems for genome editing. By optimizing the metal ion binding site and the residues involved in catalysis, it may be possible to improve the efficiency and accuracy of CRISPR-Cas nucleases in targeting specific DNA sequences for editing. Additionally, the insights into the deprotonation pathway of the nucleophilic water could guide the design of nucleases with enhanced catalytic activity and reduced off-target effects.
Furthermore, the understanding of the concerted mechanism of metal ion binding and water deprotonation could be leveraged to engineer nucleases for various biotechnological applications, such as gene therapy, DNA repair, and biocatalysis. By fine-tuning the active site architecture and metal ion coordination, researchers can create customized nucleases with improved properties for specific applications in biotechnology and medicine.