Engineering Enzymes to Develop Hormone Biosensors
By Chloe de Leon
Hormonal imbalances affect 20% of the population, meaning technology that can track these hormone levels is essential, according to fifth-year PhD student Takumi Hawes.
Hawes conducts research in the Department of Chemistry in Professor Karen Allen’s lab, where he studies and modifies enzymes so they bind with certain steroid hormones.
Hawes works with the microbial enzyme family 3-ketosteroid Δ1-dehydrogenases, or KSDHs. This particular family of enzymes is found in bacteria, fungal microbes, and archaea. Enzymes bind to molecules to facilitate chemical reactions. The bound molecule is known as the enzyme’s substrate. An enzyme’s affinity to a specific substrate is determined by the geometry and composition of its active site. Hawes’s study aims to study KSDHs from microbes and mutate them to accept different steroid hormones such as cortisol (related to stress) and progesterone and testosterone (related to fertility).
“Enzymes are experts at finding a needle in a haystack [and] essentially just being able to hone in to find the one substrate that they work on,” Hawes said.
KSDH genes are identified either from a database or by genomic screening. The DNA can then be ligated into a plasmid and grown in E. coli. After testing the KSDH against the 16 steroids, Hawes uses Google’s artificial intelligence-powered software, AlphaFold, to study the enzyme active site and propose mutations that could shift specificity to a different steroid. In this process, Hawes engineered a KSDH variant specific to testosterone even though he was trying to engineer specificity for cortisol.
James Galagan’s Biomedical Engineering lab utilizes Hawes’s research to develop biosensors that measure hormone concentrations in the body. The guiding principles for their work come from the design of glucose monitors used by diabetics to measure blood sugar. Glucometers utilize glucose oxidase, another enzyme isolated from a microbe, to continuously sample glucose in blood and measure it in real-time. Some enzymes, like glucose oxidase and KSDHs, rely on molecules or ions called cofactors to act on substrates. Although they act on different substrates, KSDHs and glucose oxidase both use their cofactors to accept electrons from their substrates and transfer those electrons to an electrode. This electric current produces a signal proportional to the substrate concentration. This continuous and accurate ability to track levels of glucose or hormones can offer valuable health insights.
Hawes explained that engineers in the Galagan lab are working towards a multiplex sensor that can use KSDH variants to simultaneously track several steroid hormone concentrations. While Hawes’s primary research focuses on discovering the complexities and characteristics of varying enzymes, he is also excited for the applications of his research in healthcare solutions.
“A big thing that I like is that there is a direct application of my work that will be used to help people,” Hawes said. “That was a really important thing for me when I started grad school and joined this project. I like this idea that with enough time, these steroid responsive biosensors can be developed, and people will be able to better monitor things like stress and fertility.”