Document Type

Honors Project

First Advisor

Dr. Rippa Sehgal

Second Advisor

Dr. Stephen Baron

Third Advisor

Dr. Ian McNeil

Degree Award Date

Fall 2025

Keywords

12α-hydroxysteroid dehydrogenase; bile acid metabolism; steroid dehydrogenase; enzymatic oxidation-reduction; NAD(P)(H)-dependent enzymes; bile acid degradation; gut microbial metabolism; enzyme kinetics; structure–function relationship

Disciplines

Biochemistry | Molecular Biology | Other Chemistry

Abstract

12α-Hydroxysteroid dehydrogenase (12α-HSDH) is an NAD(P)H-dependent oxidoreductase involved in bile acid interconversion within the human gut microbiome. Because bile acid–modifying enzymes influence host–microbe interactions and gut health, this project aimed to characterize the biochemical properties and structural features of recombinant 12α-HSDH from Methanosphaera stadtmanae. Initial attempts to generate new transformants were unsuccessful; a plasmid integrity check revealed no detectable plasmid DNA, indicating plasmid loss. Consequently, all expression was performed using a previously prepared glycerol stock, which reliably produced soluble enzyme under IPTG induction at 16 °C. A major focus of the study was purification optimization. Early FPLC-based Ni²⁺ IMAC produced low yields and inconsistent purity, whereas transitioning to gravity-flow cobalt affinity chromatography, combined with optimized wash stringency at 60 mM imidazole, yielded highly purified protein. SDS-PAGE and NanoDrop analyses confirmed substantial improvements in purity, and final concentrated samples consistently reached ~5 mg/mL, enabling structural and kinetic studies.

Purified 12α-HSDH was used for both small-angle X-ray scattering (SAXS) and crystallization trials. High-quality concentrated samples were submitted for SAXS analysis, and optimized crystallization conditions (1% PEG 400, 2.0 M ammonium sulfate, 0.1 M HEPES pH 6) generated well-formed rod-shaped crystals suitable for future X-ray diffraction. Enzyme kinetics were characterized in the reductive direction using NADPH consumption at 340 nm. Michaelis–Menten and Lineweaver–Burk analyses produced consistent kinetic parameters, including a Vmax of ~0.02 mM/min, KM of ~0.2 mM, kcat of ~8–10 s⁻¹, and catalytic efficiency of ~46.7 s⁻¹·mM⁻¹.

Overall, this work established a reliable expression system, optimized purification strategy, and produced high-quality structural samples, while quantitatively defining the reductive catalytic behavior of 12α-HSDH. These results set the stage for future mutational analysis, expanded kinetic profiling, and complete structural determination, which together will advance understanding of bile acid metabolism by archaeal members of the human gut microbiome.

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