Structure-guided engineering of human hyaluronidase PH20 for simultaneously enhanced catalytic efficiency and thermostability.

Abstract

Human hyaluronidase PH20 hydrolyzes hyaluronan, the major extracellular-matrix glycosaminoglycan, and is used to disperse subcutaneously administered biologics. Its broader use is limited by modest neutral-pH activity and poor thermostability, two properties that rarely improve together because their respective substitutions often interfere. Guided by the cryo-electron microscopy structure of PH20, two spatially separate mutation sets are designed: six second-shell substitutions near the substrate groove, and four distal stabilizing substitutions. When evaluated separately, the activity-enhanced variant AC12 exhibits a 5.4-fold higher apparent catalytic efficiency (kcat/Km) at pH 7.0 than wild-type PH20, whereas the thermostabilized variant TC1 exhibits a melting temperature 4.7 °C higher. However, the variant combining the two substitution sets retains the full thermostability gain but only part of the activity gain. Single-reversion analysis identifies one context-dependent residue whose reversion restores catalysis without stability loss. A second design round introduces consensus-guided proline substitutions at two loop positions, yielding the final combined activity- and thermostability-enhanced variant, ATC36. Relative to wild-type PH20, ATC36 exhibits a 5.5-fold higher apparent catalytic efficiency, melting and half-inactivation temperatures 17.1 °C and 16.4 °C higher, respectively, and a 9.0-fold higher purification yield. At physiological temperature (37 °C), wild-type PH20 activity falls below 50% of its initial value within 2 h, whereas ATC36 activity remains above 50% after 24 h. Beyond yielding an improved PH20 biocatalyst for subcutaneous delivery, these results show that spatial separation of function-tuning substitutions does not guarantee additivity, and that iterative combine-then-reassess back-mutation efficiently diagnoses and resolves epistatic conflicts in multiparameter enzyme engineering.

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