Abstract
Streptococcus pyogenes is an important zoonotic Gram-positive bacterium that appears in chains, without spores or flagella, and belongs to the beta-hemolytic streptococci. 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 Streptococcus pyogenes; its accumulation in response to various stress signals can inhibit bacterial pathogenicity and enhance antibiotic susceptibility. Our research focuses on the Sp-yqeK protein, which we have identified as a hydrolase that symmetrically cleaves Ap4A. The Sp-yqeK 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 Sp-yqeK, Mg(2+), and ADP complex at a resolution of 2.0 Å, providing insights into the interactions crucial for catalytic efficiency between Sp-yqeK and ADP. This complex reveals unique folding characteristics of the HD domain superfamily proteins, accommodating both ADP and Mg(2+). These components are securely embedded into the polar cavity of the yqeK protein through conserved residues (His(29), Lys(62), His(91), His(117), Asp(135), Leu(172), Phe(180), and Thr(183)), highlighting the residues responsible for Ap4A hydrolysis and Mg(2+) binding. Our research offers a deeper understanding of the hydrolysis mechanism of Ap4A and the specificity of Sp-yqeK, providing structural insights that may support future studies on antibiotic resistance in Streptococcus pyogenes and other Gram-positive bacteria.