Investigation of the Tyrosine Phosphatase Activity of Cps2D From Streptococcus suis Serotype 2.

Abstract

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen, with its capsular polysaccharide (CPS) serving as a key virulence factor. However, the regulatory mechanisms of CPS biosynthesis remain unclear. This study aimed to prepare and characterize the capsular synthesis regulatory factor Cps2D from S. suis 2 and investigate its enzymatic activity. Bioinformatics analysis using ProtParam and Alpha Fold revealed Cps2D as a hydrophilic, stable protein of 242 amino acids, with a molecular weight of 28 kDa and a pI of 8.69. It was annotated as a tyrosine protein phosphatase, primarily composed of α-helices and random coils, and structurally homologous to the protein model 3qy8.1.A, suggesting potential interactions with glycosyltransferases involved in capsule synthesis. The cps2D gene was amplified from S. suis 2 strain 05ZYH33 and cloned into the pET30a vector, and the recombinant protein was successfully purified via Ni²⁺-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot. Enzymatic characterization using p-nitrophenyl phosphate as a substrate showed that purified Cps2D exhibited phosphatase activity of 81.64 ± 0.72 U/L. The enzyme showed maximal activity at pH 10.0 and 37°C. Among the divalent metal ions tested, Mg^{2 +} produced the strongest stimulatory effect, with 5 mM Mg^{2 +} yielding the highest activity. Kinetic analysis under optimized conditions gave a K_m of 1.367 mM, V_max of 1.012 × 10^{- 2} mM min^{- 1}, K_cat of 0.4819 min^{- 1}, and K_cat/K_m of 0.3525 min^{- 1} mM^{- 1}. These results establish a soluble prokaryotic expression and purification strategy for Cps2D and provide the first biochemical evidence supporting its phosphatase activity in vitro. The purified recombinant protein will facilitate future structural and mechanistic studies of phosphorylation-dependent regulation of capsule-associated proteins in S. suis serotype 2.