# Engineering promiscuous enzymes for synthesis of biological building blocks

> **NIH NIH R35** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $400,154

## Abstract

The use of enzymes in synthesis has had an enormous impact on the development of bioactive
compounds, as they can perform transformations with unparalleled selectivity at a low cost without
using toxic solvents. However, there is a dearth of enzymes that catalyze C-C bond formation on
preparative scales. Many common enzymes from central metabolism have exquisite substrate
selectivity or rely on coupling to downstream reactions as a thermodynamic driving force, limiting
their utility. We have identified a suit of pyridoxal-phosphate (PLP) dependent enzymes that
catalyze stand-alone C-C bond forming reactions that are mechanistically distinct from their
counterparts in central metabolism. We propose mechanistic analysis of these enzymes,
encompassing structural, kinetic, and spectroscopic studies, that will reveal how these enzymes
form high-energy intermediates that are shielded from destructive interactions with solvent. This
information will enable hypothesis-driven strategies to alter and improve enzyme function. In an
allied effort, we are exploring new strategies to increase the efficiency of screening-based directed
evolution. Assaying mixtures of substrates in direct competition, followed by resolution and
quantitation of the products contain a wealth of un-tapped information. We will explore how to
maximize the information present in substrate mixtures and how to use multiplexed data to guide
evolutionary steps that are driven by either changes in total activity or by changes in specificity.
These advances in engineering will synergize with our practical efforts to evolve C-C bond forming
enzymes to perform new catalytic reactions. This research will have immediate impacts because
the enzymes will produce non-canonical amino acids (ncAAs). Nature often uses ncAAs bearing
side chain stereocenters to tune bioactivity, but the structural complexity of these molecules
makes many out of reach for standard organic chemistry. The ncAAs made here will add new and
valuable diversity to the medicinal chemistry repertoire.

## Key facts

- **NIH application ID:** 10842118
- **Project number:** 1R35GM153276-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Andrew Buller
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $400,154
- **Award type:** 1
- **Project period:** 2024-08-15 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10842118

## Citation

> US National Institutes of Health, RePORTER application 10842118, Engineering promiscuous enzymes for synthesis of biological building blocks (1R35GM153276-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10842118. Licensed CC0.

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