Abstract
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.
Highlights
Hyaluronan (HA),3 a linear polysaccharide found in the extracellular matrix of most tissues and body fluids of vertebrates, is enzymatically degraded by hyaluronidases [1]
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 hyaluronidase 1 (HYAL1) to be crucial for substrate hydrolysis
HA hydrolysis is achieved by a pair of acidic amino acids via a retaining double displacement mechanism and a substrate-assisted catalysis, in which the carbonyl oxygen of the N-acetyl group of the cleaved HA subunit acts as the catalytic nucleophile [7]
Summary
Designed Human Serum Hyaluronidase 1 Variant, HYAL1⌬L, Exhibits Activity up to pH 5.9. 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. We present computational and experimental data on the replacement of a loop region located at the end of the substrate binding groove yielding a variant hyaluronidase with an altered pH profile
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