Designed Human Serum Hyaluronidase1Variant,HYAL1 L ,

Hyaluronidasesfromdiversespeciesandsourceshavediffer-ent pH optima. Distinct mechanisms with regard to dynamicstructuralchanges,whichcontrolhyaluronidaseactivityatvary-ing pH, are unknown. Human serum hyaluronidase 1 (HYAL1)is active solely below pH 5.1. Here we report the design of aHYAL1 variant that degrades hyaluronan up to pH 5.9. Besideshighlyconservedresiduesincloseproximityoftheactivesiteofmost hyaluronidases, we identified a bulky loop formationlocated at the end of the substrate binding crevice of HYAL1 tobecrucialforsubstratehydrolysis.Thestretchbetweencysteineresidues 207 and 221, which normally contains 13 amino acids,couldbereplacedbyatetrapeptidesequenceofalternatinggly-cine serine residues, thereby yielding an active enzyme with anextended binding cleft. This variant exhibited hyaluronan deg-radation at elevated pH. This is indicative for appropriate sub-strate binding and proper positioning being decisively affectedby sites far off from the active center.