Aromatic interactions surrounding the Asp-X-Glu motif stabilize the active site of HYAL1 and PH20

Hyaluronidases degrade hyaluronic acid and participate in physiological processes, including tissue remodeling and fertilization. Vertebrate and venom hyaluronidases share a conserved Asp-X-Glu catalytic motif in the β4 loop (X = Trp or Phe). Here, we investigated HYAL1, a lysosomal enzyme, and compared it with PH20, a sperm surface hyaluronidase, to elucidate how conserved aromatic residues surrounding this motif stabilize the active site, particularly under acidic conditions. In HYAL1, Tyr82 (β3' loop), Trp130 (X residue), and Trp133 form an aromatic cluster adjacent to the active site. Substitutions at these positions reduced activity at pH 4, indicating that this cluster is required to maintain a catalytically competent conformation, with Tyr82 stabilizing the β3'-β4 loops through hydrogen bonding and Trp130 and Trp133 providing aromatic stabilization, with Trp133 playing a dominant role. A Phe-Phe interaction, analogous to that in insect venom hyaluronidases (Y82F/W130F), partially rescued activity and active-site stability compared with single substitutions. In PH20, corresponding substitutions caused greater activity losses at pH 4, indicating increased structural sensitivity to acidic conditions despite its broad pH range. pH-dependent ANS fluorescence revealed more pronounced acid-induced conformational changes at pH 4 than at pH 7 in both enzymes. Molecular dynamics simulations indicated that HYAL1, PH20, and bee venom hyaluronidase retain a rigid catalytic core, with differences arising primarily from loop and domain flexibility. These findings show that aromatic interactions surrounding the Asp-X-Glu motif stabilize the active site, whereas Phe-Phe interactions represent an alternative active-site stabilization strategy in insect venom hyaluronidases.