Bacteriolytic enzymes are promising antimicrobial agents for developing new-generation drugs. Recently, we have isolated a β-lytic protease (BlpLc) from the culture liquid of Lysobacter capsici VKM B-2533^T. This BlpLc possesses a valuable property, not described for β-lytic proteases (Blps) earlier, of hydrolyzing living cells of Staphylococcus aureus 55 MRSA clinical isolate. This work phylogenetically characterized the BlpLc and investigated its properties. Analysis revealed a variability of pre-/pro-parts of Blp precursors. The mature BlpLc is the closest to the earlier annotated but not isolated Blp from Lysobacter sp. Root690. The biochemical characterization found conditions for the BlpLc general bacteriolytic activity relative to autoclaved S. aureus 209P cells to differ from that of earlier isolated Blp. Unexpected was the effect of serine (phenylmethylsulfonyl fluoride (PMSF)) and cysteine (p-chloromercuribenzoate (p-CMB)) protease inhibitors on BlpLc bacteriolytic and proteolytic activities. The specificity of BlpLc proteolytic action relative to hemoglobin, elastin, gelatin, collagen, azofibrin, myoglobin, ovalbumin, and ovamucoid was found. New types of peptide bonds—Gly-X, Ser-X, Lys-X, Ala-X, Val-X, Glu-X, and Phe-X—hydrolyzed by the enzyme in protein substrates were first revealed using MALDI-TOF. Turbidimetrically, the BlpLc was found to lyze living cells of S. aureus 209P, Micrococcus luteus B1819, and M. roseus B1236, which is important for expanding the enzyme’s applied properties.

β-Lytic protease (Blp) and protease L5 are enzymes from Lysobacter bacteria with distinct proteolytic and bacteriolytic activities. To characterize their substrate specificity, we employed liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis following hydrolysis of fractionated protein mixtures. Heatmaps and sequence logos revealed a pronounced specificity of Blp towards glycine and lysine residues, while L5 preferentially cleaved non-polar residues such as methionine, phenylalanine, and leucine. Notably, proline was frequently observed at the P2 position in L5 substrates. Comparative analysis with trypsin revealed that L5 generated significantly shorter peptides, whereas Blp produced fragments similar in length to tryptic peptides. These findings indicate different cleavage preferences and suggest potential applications for these enzymes in proteomic analysis.

The high physiological activity of steroid hormones requires multilevel regulation of their biosynthesis. Cytosolic sulfotransferases, specifically SULT2A1 and SULT1E1, convert steroid precursors into mobile sulfated depot forms, diverting them from key steroidogenic cytochromes P450. Given the previously demonstrated CYP17A1–SULT interactions, this study aims to verify the hypothesis that CYP21A2 also interacts with SULTs to co-regulate steroid biosynthesis. Using surface plasmon resonance (SPR) biosensing, in vitro enzymatic activity assays, and in silico molecular modeling, we investigated the interplay between CYP21A2 and SULT2A1/1E1. SPR analysis revealed direct, high-affinity protein–protein interactions in the nanomolar range. Thermodynamic assessments indicated that the CYP21A2–SULT1E1 complex is entropy-driven, contrasting with the enthalpy-driven CYP17A1–SULT1E1 interaction. Enzymatic assays demonstrated reciprocal modulatory effects: CYP21A2 exerted a stimulatory effect on pregnenolone sulfation by SULTs, whereas SULT2A1 moderately inhibited CYP21A2 activity. Molecular modeling of binary complexes predicted structural interfaces and continuous intraprotein channels connecting the enzymes' active sites. These findings suggest that CYP–SULT interactions contribute to the fine-tuning of steroid hormone biosynthesis. We propose that these enzymes participate in a highly organized multiprotein network, acting as metabolic switches that dictate the balance between downstream corticosteroid pathways and structural sequestration into sulfated pools.

The high physiological activity of steroid hormones requires multilevel regulation of their biosynthesis. Cytosolic sulfotransferases, specifically SULT2A1 and SULT1E1, convert steroid precursors into mobile sulfated depot forms, diverting them from key steroidogenic cytochromes P450. Given the previously demonstrated CYP17A1–SULT interactions, this study aims to verify the hypothesis that CYP21A2 can interact with SULTs and thereby potentially contribute to the regulation of steroid metabolism. Using surface plasmon resonance (SPR) biosensing, in vitro enzymatic activity assays, and in silico molecular modeling, we investigated the interplay between CYP21A2 and SULT2A1/1E1. SPR analysis revealed high-affinity binding between purified recombinant CYP21A2 and SULT2A1/SULT1E1 in vitro in the nanomolar range (9.5 × 10⁻⁹ M for SULT2A1 and 1.15 × 10⁻⁸ M for SULT1E1). Thermodynamic assessments indicated that the CYP21A2–SULT1E1 complex is entropy-driven, contrasting with the enthalpy-driven CYP17A1–SULT1E1 interaction. Enzymatic assays demonstrated reciprocal modulatory effects: CYP21A2 exerted a stimulatory effect on pregnenolone sulfation by SULTs, whereas SULT2A1 moderately inhibited CYP21A2 activity. Molecular modeling of binary complexes predicted structural interfaces and continuous intraprotein channels connecting the enzymes’ active sites. These findings suggest that CYP–SULT interactions may contribute to the fine-tuning of steroid hormone biosynthesis. These findings raise the possibility that CYP–SULT interactions may form part of a broader regulatory network, acting as metabolic switches that dictate the balance between downstream corticosteroid pathways and structural sequestration into sulfated pools.