# Biophysical constraints on evolution of enzyme specificity

> **NIH NIH R01** · TEXAS A&M AGRILIFE RESEARCH · 2020 · $276,909

## Abstract

Project Summary
 Designing enzymes that are as efficient as natural enzymes is very difficult, showing that we
understand too little about the biophysical constraints that limit evolutionary routes to new functions. In-depth
studies about the biophysical constraints on protein evolution have been limited to a few model systems.
These studies indicate that epistasis, which occurs when mutations have different effects in different
sequence contexts, is common, but its biophysical basis and pervasiveness are not well studied. Promiscuity,
which is the coincidental ability to carry out a reaction that is not a biological function, also plays a role by
providing the raw material for natural selection to evolve new biological functions. Underlying promiscuity and
epistasis is protein biophysics: structure, stability, dynamics, and enzymatic mechanism. The goal of this
proposal is to illuminate the roles of promiscuity and epistasis in protein evolution by comparing biophysical
constraints on promiscuous enzymes to those of highly specific enzymes. We established the N-succinylamino
acid racemase (NSAR)/o-succinylbenzoate synthase (OSBS) subfamily as one of the best models for
determining the role of promiscuity in enzyme evolution. NSAR activity evolved from an ancestral OSBS, and
many enzymes are catalytically promiscuous for both activities. Those that have NSAR activity also exhibit
substrate promiscuity, preferring hydrophobic N-succinylamino acids but having weak activity with other side
chains. This proposal hypothesizes that promiscuity is correlated with the nature and extent of biophysical
constraints on mutations that are required to evolve new activities. Our aims are to 1) Define the biophysical
constraints on evolution of NSAR activity in a highly specific OSBS enzyme. We will test the hypothesis
that several highly specific OSBSs will evolve NSAR activity by different routes due to epistasis and other
biophysical constraints; 2) Compare biophysical constraints on promiscuous NSAR/OSBS enzymes and
highly specific OSBS enzymes by testing the hypothesis that changing the N-succinylamino acid preference
of promiscuous NSAR/OSBS enzymes will be more feasible than changing substrate preference of highly
specific OSBSs; and 3) Develop a general approach to identify epistatic interactions. These aims will be a
significant step toward determining how structure, stability, dynamics and catalytic mechanism affect the
evolution of new enzyme activities. Comparing promiscuous and highly specific enzymes will clarify the role of
promiscuity. Biophysical analysis will reveal epistatic mechanisms, especially effects of mutations distant from
the active site. We will leverage this data with a massively parallel analysis of mutation phenotypes to develop
a general approach to identify epistatic interactions. We will use this approach to refine protein engineering
strategies, steering mutagenesis toward epistatic sites that need to be simultaneously optimiz...

## Key facts

- **NIH application ID:** 9952389
- **Project number:** 5R01GM124409-03
- **Recipient organization:** TEXAS A&M AGRILIFE RESEARCH
- **Principal Investigator:** MARGARET E GLASNER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $276,909
- **Award type:** 5
- **Project period:** 2018-08-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9952389

## Citation

> US National Institutes of Health, RePORTER application 9952389, Biophysical constraints on evolution of enzyme specificity (5R01GM124409-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9952389. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*