Designed human serum hyaluronidase 1 variant, HYAL1DeltaL, exhibits activity up to pH 5.9.
Hyaluronidases from diverse species and sources have different pH optima. Distinct mechanisms with regard to dynamic structural changes, which control hyaluronidase activity at varying pH, are unknown. Human serum hyaluronidase 1 (HYAL1) is active solely below pH 5.1. Here we report the design of a HYAL1 variant that degrades hyaluronan up to pH 5.9. Besides highly conserved residues in close proximity of the active site of most hyaluronidases, we identified a bulky loop formation located at the end of the substrate binding crevice of HYAL1 to be crucial for substrate hydrolysis. The stretch between cysteine residues 207 and 221, which normally contains 13 amino acids, could be replaced by a tetrapeptide sequence of alternating glycine serine residues, thereby yielding an active enzyme with an extended binding cleft. This variant exhibited hyaluronan degradation at elevated pH. This is indicative for appropriate substrate binding and proper positioning being decisively affected by sites far off from the active center.
Reitinger
Stephan
S
Extracellular Matrix Research Group, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria.
Müllegger
Johannes
J
Greiderer
Brigitte
B
Nielsen
Jens Erik
JE
Lepperdinger
Günter
G
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D005260:Female; D014644:Genetic Variation; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D014982:Xenopus laevis
D050505:Mutant Proteins; D011994:Recombinant Proteins; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase