Investigation of the Structure and Functional Activity of the YqeK Protein in Streptococcus pyogenes with High Efficiency in Hydrolyzing Ap4A

<i>Streptococcus pyogenes</i> is an important zoonotic Gram-positive bacterium that appears in chains, without spores or flagella, and belongs to the <i>beta-hemolytic streptococci</i>. It can be transmitted through droplets or contact, with the preferred antibiotics being penicillin, erythromycin, or cephalosporins. However, the misuse of these drugs has led to antibiotic resistance, posing a significant threat to both human and animal health. Studying resistance genes encoding proteins is crucial for mitigating the emergence of resistant strains and improving treatment outcomes. Interestingly, a dinucleotide known as diadenosine tetraphosphate (Ap4A) exists in <i>Streptococcus pyogenes</i>; its accumulation in response to various stress signals can inhibit bacterial pathogenicity and enhance antibiotic susceptibility. Our research focuses on the <i>Sp-yqeK</i> protein, which we have identified as a hydrolase that symmetrically cleaves Ap4A. The <i>Sp-yqeK</i> protein effectively cleaves Ap4A, producing adenosine diphosphate (ADP) molecules. Results indicate that this enzyme exhibits optimal activity at pH 7.0 and a temperature of 45 °C. Furthermore, we determined the crystal structure of the <i>Sp-yqeK</i>, Mg²⁺, and ADP complex at a resolution of 2.0 Å, providing insights into the interactions crucial for catalytic efficiency between <i>Sp-yqeK</i> and ADP. This complex reveals unique folding characteristics of the HD domain superfamily proteins, accommodating both ADP and Mg²⁺. These components are securely embedded into the polar cavity of the yqeK protein through conserved residues (His²⁹, Lys⁶², His⁹¹, His¹¹⁷, Asp¹³⁵, Leu¹⁷², Phe¹⁸⁰, and Thr¹⁸³), highlighting the residues responsible for Ap4A hydrolysis and Mg²⁺ binding. Our research offers a deeper understanding of the hydrolysis mechanism of Ap4A and the specificity of <i>Sp-yqeK</i>, providing structural insights that may support future studies on antibiotic resistance in <i>Streptococcus pyogenes</i> and other Gram-positive bacteria.