Dissecting enzyme function at scale using synergistic advances in microfluidics and genetic code expansion

PROJECT SUMMARY.
Noncanonical amino acids (ncAAs) have myriad valuable applications in the biochemical and biophysical
sciences. Their site-specific incorporation into proteins of interest can directly install systematically perturbed
residues, sensitive biophysical probes, bio-orthogonal handles, and post-translational modifications (PTMs) at
positions of interest. While promising, these applications have been greatly limited by costly materials and labor-
intensive, low-yielding preparations. To realize the full potential of ncAAs, I will leverage the recently developed
high-throughput microfluidic enzyme kinetics (HT-MEK) platform from the Fordyce and Herschlag laboratories
at Stanford University to enable the parallel expression, purification, and quantitative assay of >1,000 ncAA-
harboring protein variants on a single microfluidic device. With this approach, it will become feasible and routine
to collect >10,000 gold-standard biochemical measurements of ncAA-containing proteins while using less
material and effort than is typically required to collect a single such measurement.
To illustrate the power and utility of this technique, I will first apply it towards understanding the catalytic
mechanisms governing proton transfer at carbon in the model system alanine racemase (AlaR), an important
pyridoxal 5’-phosphate (PLP)-dependent enzyme involved in cell-wall biosynthesis. PLP-dependent enzymes
account for 4% of all classified enzymatic activities and ~1.5% of prokaryotic reading frames, and they are
increasingly important in biotechnology. Although we have a reasonable understanding of how the small-
molecule cofactor itself can influence catalysis, the specific contributions of the protein scaffold remain
speculative, qualitative, or both. Previous studies that have used traditional site-directed mutagenesis—altering
many properties simultaneously—and only examined a handful of variants have failed to deliver a unified view
of how this enzyme achieves its catalytic proficiency. Here, I will use ncAAs on the HT-MEK device to
systematically and precisely perturb the electrostatic properties of critical catalytic residues in the active site of
AlaR—leaving other steric properties largely unaltered—across 96 different enzyme variants. Specifically, I will
investigate how interactions in the active site act together to optimize this difficult proton transfer to: (1) be highly
efficient at neutral pH; and (2) achieve an exquisite 106:1 regioselectivity among competing pathways for
reprotonation of the reactive intermediate.
The new training that I obtain from this project will greatly and uniquely expand my skillset at the interface of
biocatalysis and mechanistic enzymology, leaving me poised to achieve my long-term goal of creating new
enzymes to address enduring and emergent challenges in the biological and chemical sciences. More broadly,
the development of reliable methods for the quantitative, high-throughput assay of hundreds of ncAA-harboring
proteins is expected to have far-reaching impacts in all areas of biochemical and biophysical research with
significant applied and therapeutic relevance.

Grant Number: 5F32GM156066-02
NIH Institute/Center: NIH
Principal Investigator: Patrick Almhjell