In the current fight against pathogenic microorganisms, the problems of multiple antibiotic resistance and the ability to form biofilms were identified. Therefore, searching new compounds providing antibacterial activity represent an important and urgent task. It has been shown that the growth of pathogenic Klebsiella bacteria is suppressed when co-cultured with lactobacilli. At the same time, releasing a number of lactobacillus enzymes into the external environment was noted. The proteomic analysis of the released proteins has shown that lactobacilli secreted three types of proteins in the response to the Klebsiella action: bacterial cell wall hydrolases, nucleic acid hydrolases, and enzymes of cellular metabolism. Cysteine synthase A was one of these enzymes [1].

O-acetylserine sulfhydrolase-A, also known as cysteine synthase A (CysK) (EC:2.5.1.47), is a pyridoxal 5′-phosphate (PLP) dependent enzyme from the class of transferases that catalyzes the transfer of a HS− group from sulfide to O-acetyl-L-serine to form L-cysteine (Cys) in bacteria and plants (Fig. 1). This is the final stage of cysteine synthesis, which is a β-substitution reaction. In the first step, O-acetyl-L-serine forms an external aldimine, which undergoes β-elimination of the acetoxy group. This leads to the appearance of the Schiff aminoacrylate base, further attacked by sulfide to form the external aldimine cysteine, which is eventually released to regenerate the internal aldimine [2]. This is the predominant pathway for incorporating reduced sulfur into organic compounds in bacteria, archaea, and plants, as well as an additional pathway for fungi [[3], [4], [5], [6]]. The prospect of using cysteine synthase for cysteine producing [7] claims its thorough studying. This requires a method development for determining its enzymatic activity. Cysteine synthase is an important enzyme in the vital activity of many bacteria, including pathogenic ones, as well as plants [8,9]. Currently, the search for specific inhibitors of this enzyme in pathogens is actively underway providing possibility for creating drugs and herbicides on their basis.

To the date, the most popular method for studying the enzymatic activity of CysK [[10], [11], [12], [13]] was developed by M.A. Gaintode in 1967 [14] and adapted in [15]. It is based on the formation of a colored product (detected at 560 nm) after the interaction of cysteine and ninhydrin in an acidic environment at high temperature. This method has a number of disadvantages, such as poor selectivity, sensitivity, and accuracy. Also, precipitation occurs for high concentrations of sulfide as one of the enzyme substrates, followed by the absorption of the colored product in some buffer systems.

Thus, the most popular spectroscopic methods for studying CysK enzymatic activity have some problems, including the proximity of the absorption maxima for the substrates and products of the enzymatic reaction. This obviously requires their chromatographic separation. To the date, there are only a few works utilizing chromatography for studying the enzymatic activity of this enzyme or similar ones. All the existing ones use only the reversed-phase high-performance liquid chromatography (RP HPLC) requiring preliminary derivatization of the polar product.

Cysteine is a sulfur-containing amino acid, which is also classified as a thiol. Thiols are categorized as challenging group of analytes because they are prone to oxidation and do not have readily detectable and distinguishable spectroscopic properties [16]. Among the chromatographic methods for its determination, RP HPLC with chemical derivatization is a widespread approach in order both to stabilize the analytes and increase their sensitivity [[17], [18], [19], [20]]. In [21] an HPLC method based on derivatization of the thiol group of cysteine with monobromobimane and formation of a fluorescent adduct was applied for the most similar task on studying cysteine synthase activity. The separation of the bimane derivative was carried out by RP HPLC where cysteine derivative was separated from two substrates in 24 min including re-equilibration time. Besides, a time-consuming derivatization procedure and a selective fluorescence detector was required. Quite a similar task was accomplished in [22] for studying the related CysM-encoded enzyme S-sulfocysteine synthase by quantification of the total cysteine and glutathione contents in RP HPLC mode with the same derivatization agent. Separation time was 15 min with using gradient elution including re-equilibration step. However, it required even more sophisticated and less available mass spectrometric detection. The approach to determining the activity of cystathionine β-synthase and γ-cystathionase was applied in [23], where cysteine was the substrate. It was performed by RP HPLC with UV-detection and 1-fluoro-2,4-dinitrobenzene as derivatizing agent. This analysis took 85 min in gradient elution mode including re-equilibration time. It was crucial to separate the products from cysteine, co-substrate, and by-products of the derivatization reaction. Thus, the required derivatization step is the main problem in RP HPLC mode for such a task. Also, scientists in the field of enzymology mainly refer to the old methods [[24], [25], [26]] with some modifications, which leads to significant analysis time and does not satisfy the modern pace of research.

Hydrophilic interaction liquid chromatography (HILIC) is a promising method for direct determination of highly polar cysteine [27]. The undoubted advantage of HILIC is the absence of derivatization step for amino acids [[28], [29], [30], [31]]. Also, this method can provide an alternative and increased selectivity as compared to RP HPLC, which is extremely necessary for separating the target complex samples. It is also a convenient tool for studying the kinetics of various enzymes because of the usual polarity of the substrates used and the products obtained. However, the utilization of HILIC is only now gaining popularity in the field of enzymology [[32], [33], [34], [35], [36], [37], [38]], but mainly with the application of sophisticated mass spectrometric detection, which requires an advanced operator [[33], [34], [35],37,38]. We have recently developed a novel HILIC method for fast nucleosides and corresponding nitrogenous bases separation and determination to assess enzymatic activity of the ribonucleoside hydrolase C [39,40]. Using HILIC and the developed column we reduced the total time for obtaining the plot for the enzymatic reaction rate by 5 times (from 40 to 8 h for the whole analysis) as compared to RP HPLC method. As soon as enzymological tasks often require hundreds and thousands of analyses, it is crucial to emphasize the importance of reducing the analysis time for singular injection. Thus, the existing methods for the determination of such an enzyme reaction rate constants require additional chromatographic separation by RP HPLC, which is time- and labor-consuming due to derivatization of cysteine.

Herein, we describe the development of a HILIC method as a modern and available alternative to the above-mentioned spectroscopic and RP-HPLC methods for the determination of cysteine and the study of the enzymatic activity of CysK using UV-detection and novel laboratory-developed hydrophilic stationary phases synthesized by the multicomponent Ugi reaction. Special attention is paid to choosing elution conditions due to the presence of interfering components of the enzymatic reaction and studying their retention mechanisms. Developing a method using HILIC and special stationary phases is the key to a much faster and less labor-intensive direct analysis for obtaining the rate of the CysK enzymatic reaction employing simple HPLC systems with isocratic elution and UV-detection without any derivatization step.