Substitution of acidic residues near the catalytic Glu131 leads to human HYAL1 activity at neutral pH via charge-charge interactions.

Human hyaluronidase 1 (HYAL1) and PH20 play vital roles in degrading hyaluronic acids through the substrate-assisted double displacement mechanism. While HYAL1, a lysosomal enzyme, functions optimally under acidic conditions, PH20, a sperm surface hyaluronidase, displays a broader pH range, from acidic to neutral. Our objective was to extend HYAL1's pH range towards neutral pH by introducing repulsive charge-charge interactions involving the catalytic Glu131, increasing its pKa as the proton donor. Substituting individual acidic residues in the ß3-loop (S77D), ß3'-ß3″ hairpin (T86D and P87E), and at Ala132 (A132D and A132E) enabled HYAL1 to demonstrate enzyme activity at pH 7, with the mutants S77D, P87E, and A132E showing the highest activity in the substrate gel assay. However, double and triple substitutions, including S77D/T86D/A132E as found in the PH20 configuration, did not result in enhanced activity compared to single substitutions. Conversely, PH20 mutants with non-acidic substitutions, such as D94S in the ß3-loop and D103T in the ß3'-ß3″ hairpin, significantly reduced activity within the pH range of 4 to 7. However, the PH20 mutant E149A, reciprocally substituted compared to A132E in HYAL1, exhibited activity similar to PH20 wild-type (WT) at pH 7. In a turbidimetric assay, HYAL1 mutants with single acidic substitutions exhibited activity similar to that of PH20 WT at pH 7. These results suggest that substituting acidic residues near Glu131 results in HYAL1 activity at neutral pH through electrostatic repulsion. This study highlights the significance of charge-charge interactions in both HYAL1 and PH20 in regulating the pH-dependent activity of hyaluronidases.

Tyr76 is essential for the cold adaptation of a class II glutaredoxin 4 with a heat-labile structure from the Arctic bacterium Sphingomonas sp.

Glutaredoxins (Grxs) are small proteins that share a well-conserved thioredoxin (Trx)-fold and participate in many biological processes. This study examined the cold adaptation mechanism of a Fe-S cluster binding class II Grx4 (SpGrx4) from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621. Three polar residues close to the cis-proline residue (P73) of SpGrx4 form a hydrogen bond network (Q74-S67-Y76) with the cis-proline loop main chain. The hydroxyl group of S67 or Y76 or both is replaced in similar Grxs depending on the temperature of the habitat. Mutants with reduced hydrogen bonds (S67A, Q74A, Y76F, and S67A/Y76W) were more susceptible to urea-induced unfolding and more flexible than the wild-type (WT). By contrast, Y76W, with a bulky indole group, was the most stable. These mutants showed higher melting temperatures than WT as a consequence of increased hydrophobic interactions. These results suggest that the tyrosine residue, Y76, is preferred for the cold adaptation of SpGrx4 with a heat-labile structure despite the rigid cis-proline loop, due to hydrogen bond formation. An aromatic residue on ß3 (cis-proline plus3) modulates the stability-flexibility of the cis-proline loop for temperature adaptation of prokaryotic class II Grx4 members via hydrogen bonds and hydrophobic interactions.