About This Project

Antibiotic resistance is rising, but nature still holds secrets to new treatments. I am studying AprL (aminotransferase encoded in the apramycin biosynthetic gene cluster), a key enzyme in apramycin biosynthesis. This project focuses on expressing and purifying AprL and understanding how it binds its essential cofactor, pyridoxal 5'-phosphate (PLP). I will uncover how this enzyme works with your support, paving the way for better antibiotic design and a stronger fight against deadly superbugs.

Ask the Scientists

What is the context of this research?

Many antibiotics we rely on today come from natural compounds made by bacteria. One such antibiotic is apramycin, which fights tough infections in animals, including humans. To make apramycin, bacteria use special enzymes to build their complex structure step-by-step. One important enzyme in this process is called AprL. AprL helps add an important chemical group needed for apramycin's activity. However, AprL is tricky to study because it requires a helper molecule called pyridoxal 5'-phosphate (PLP) that acts as a cofactor, allowing the enzyme to transfer amino groups during the biosynthetic process. Without PLP, the enzyme cannot function properly. Understanding how AprL binds PLP and functions is the first step toward potentially improving apramycin or creating new antibiotics inspired by it.

What is the significance of this project?

Antibiotic resistance is a growing global health threat that was directly responsible for 1.27 million deaths globally in 2019, making it harder to treat infections. Finding new or improved antibiotics is critical. By studying AprL, we explore a unique enzyme involved in apramycin production, a promising antibiotic that works against resistant bacteria. This project uncovers how AprL binds its helper molecule and functions, information that can guide future engineering of better antibiotics. Also, by overcoming the challenges in producing and studying AprL, we build the foundation for deeper research into this enzyme and related proteins. This foundational work will enable further studies on AprL's structure and function, advancing our understanding of antibiotic biosynthesis and opening doors to new therapeutic developments.

What are the goals of the project?

The main goal is to produce and purify the AprL enzyme from Streptoalloteichus hindustanus (MW ~42 kDa), retaining its essential PLP cofactor for structural studies. AprL will be cloned into pET-28a(+) and expressed in E. coli BL21(DE3) using ZYM-5052 auto-induction medium with a C-terminal His₆-tag. To maintain critical PLP binding, media and purification buffers will be supplemented with 0.1 mM pyridoxal 5'-phosphate. The enzyme will be purified using Ni-NTA affinity chromatography in HEPES/phosphate buffers, followed by ultra-centrifugation for concentration and size-exclusion chromatography for homogeneity. PLP binding will be verified using UV-Visible spectroscopy (characteristic 420 nm peak) and protein quantification via A₂₈₀. The primary objective is to determine the enzyme's 3D structure using X-ray crystallography and/or cryo-EM to understand PLP binding architecture. A key milestone is presenting findings at a scientific conference for expert feedback.