# Evolution of enzyme function, mechanism, dynamics, and structure viewed at atomic resolution

> **NIH NIH R01** · BRANDEIS UNIVERSITY · 2024 · $352,310

## Abstract

Project Summary
Most biochemical reactions take from hundreds to billions of years to occur spontaneously. However, life depends
on highly organized networks of catalyzed chemical reactions that proceed not only rapidly, but specifically and
with high fidelity. Biological catalysts are enzymes, complicated molecular nanomachines that massively
accelerate reactions by positioning specific substrate molecules with such precision that they are compelled to
react. The molecular mechanism by which an enzyme executes this remarkable feat involves an exquisitely
orchestrated sequence of steps. The structures, mechanisms, and functions of enzymes are all products of
millions of years of evolution. Yet despite their fundamental biological importance, we have only a rudimentary
understanding of the atomistic basis of the evolutionary changes that create novel enzymes.
In this project, we will fully elucidate, at an atomistic level of description, the biophysical principles that underlie
the evolutionary changes in structure, dynamics, and mechanism producing novel enzymatic functions. We will
resurrect entire evolutionary lineages of ancestral enzymes, solve their structures, characterize their dynamics,
and determine their kinetic mechanisms, all correlated with the functional changes observed along these
evolutionary trajectories. While we are aggressively pursuing multiple systems to maximize success, our main
model system is the malate and lactate dehydrogenase (M/LDH) superfamily. Both enzymes are found in the
core metabolism of nearly every organism on the planet. M/LDHs are homologous enzymes that share a fold and
catalytic mechanism yet can possess extraordinarily strict specificity for their substrates. The evolution of this
family is marked by many important functional innovations, including (1) sharp alterations in substrate
specificity, (2) changes in catalytic rate, (3) gain of allosteric control by small effector molecules, (4) acquisition
of thermophilic, cryophilic, halophilic, and alkalophilic stability, and (5) the evolution of multimerization via new
protein-protein interfaces. Many of these novelties are convergent, having evolved several times independently.
How do complicated, highly orchestrated kinetic mechanisms evolve? How do substitutions far from the active
site affect activity? What is the molecular basis of epistasis? Does specificity increase during evolution? Were the
ancestors of M/LDHs promiscuous? By answering these questions, we will provide the first fine-grained
description of how enzyme structures and kinetic mechanisms constrain and channel genetic evolutionary
processes. The M/LDH superfamily is a classic, well-characterized system, with a common kinetic mechanism
and cofactor. Hence, the resulting evolutionary insights will apply broadly to other enzymes and may transform
our understanding of how enzymes can be rationally engineered.

## Key facts

- **NIH application ID:** 10878493
- **Project number:** 2R01GM096053-09
- **Recipient organization:** BRANDEIS UNIVERSITY
- **Principal Investigator:** Douglas Lowell Theobald
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $352,310
- **Award type:** 2
- **Project period:** 2011-07-01 → 2028-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10878493

## Citation

> US National Institutes of Health, RePORTER application 10878493, Evolution of enzyme function, mechanism, dynamics, and structure viewed at atomic resolution (2R01GM096053-09). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10878493. Licensed CC0.

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