# De Novo Mini-Metalloenyzmes with Hydrolase Activity

> **NIH NIH R15** · GETTYSBURG COLLEGE · 2022 · $382,063

## Abstract

PROJECT SUMMARY
The major goal of the proposed work is to create, characterize, and optimize de novo binuclear mini-
metalloenzymes for hydrolase activity. Hydrolytic cleavage, or the breaking apart of a molecule by the
addition of water, is an essential biochemical reaction that governs cellular processes across all life
kingdoms. Several rare genetic diseases come from hydrolase malfunctions, making them important to
understand. Metal ions are frequently used to catalyze these reactions due to their ability to strongly
polarize the O–H bond in water even at neutral pH. A small artificial enzyme called DFsc, which
contains two metal ions in the active site, is capable of hydrolyzing small molecules and larger DNA
substrates. Activity was observed with zinc, which is commonly used by nature, and titanium, which has
no known natural function. This project seeks to (1) understand the structural and electronic factors that
contribute to hydrolase activity in the DFsc system and (2) develop additional mini-metalloenzymes with
metals underutilized by nature for environmentally-friendly and catalytically-efficient hydrolytic cleavage.
To accomplish these goals, we will design, produce, and comprehensively characterize a series of
DFsc proteins whose metal-coordinating amino acids are systematically varied. This library of proteins
will be screened for hydrolase activity using DNAse and phosphatase assays and a variety of metal
ions. Successful completion of these aims will result in a robust model system that will provide an
understanding of the structure-activity relationships in natural and artificial metallohydrolases.

## Key facts

- **NIH application ID:** 10359516
- **Project number:** 1R15GM144906-01
- **Recipient organization:** GETTYSBURG COLLEGE
- **Principal Investigator:** Katherine Michelle Buettner
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $382,063
- **Award type:** 1
- **Project period:** 2022-01-01 → 2025-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10359516

## Citation

> US National Institutes of Health, RePORTER application 10359516, De Novo Mini-Metalloenyzmes with Hydrolase Activity (1R15GM144906-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10359516. Licensed CC0.

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