Development of hydrophilic interaction liquid chromatography method for determining enzymatic activity of cysteine synthase A

The majority of enzymological tasks are targeted at determining polar compounds representing substrates, products, related substances of enzymatic reactions, etc. Currently, they are still solved either by reversed-phase HPLC requiring preliminary derivatization step for increasing hydrophobicity, or even spectroscopy remaining non-selective toward the related sample compounds. Hydrophilic interaction liquid chromatography (HILIC) method for direct determination of polar analytes provides promising solutions for the modern biochemical tasks with much easier and faster analysis for hundreds and thousands of samples. The main focus in such investigations is choosing conditions for separating complex mixtures of diverse polar compounds which could be challenging in many cases. In this work, a simple and rapid HILIC method for determining enzymatic activity of Cysteine synthase A (CysK, EC 2.5.1.47) is developed. CysK is a pyridoxal-5'-phosphate phosphate dependent enzyme that catalyzes the formation of L-cysteine and acetate from O-acetyl-L-serine and sulfide. This reaction represents a critical step in the sulfur assimilation pathway, which plays a vital role in maintaining sulfur homeostasis and supporting cellular functions such as protein synthesis, antioxidant defense, and detoxification. CysK is one of the enzymes secreted by Lactobacilli while co-cultivating with Klebsiella, which can be used for cysteine synthesis and is also important for microbial vital activity. We obtained a new recombinant CysK from Limosilactobacillus reuteri LR1 by gene cloning and expression in E.coli cells. HILIC method development for determining the enzyme activity represented a challenge of separating two substrates, two products, and a buffer component in one sample. This task was solved by choosing both the column and elution conditions. Specific amide functionalized silica-based stationary phase obtained via the Ugi reaction was chosen among the three laboratory-designed HILIC columns. The impact of ion-exchange into the retention mechanism for the main components of the enzymatic reaction was assessed for determining separation conditions on the chosen column. The developed method allowed separating cysteine from all other peaks within 11 min under isocratic elution conditions. Limits of detection and quantification were 2 and 7 μM, respectively. Using the developed method, the reaction rate of CysK obtained from Limosilactobacillus reuteri LR1 was determined for the first time. The primary benefits of this method are rapidity of the analysis, direct determination avoiding derivatization step, and utilizing simple HPLC instrumentation with neither gradient elution nor advanced detection systems. © 2025