Expanding the Catalytic Promiscuity of Heparinase III from Pedobacter heparinus

Abstract

Glycosaminoglycans (GAG) lyases are useful biocatalysts for the preparation of oligosaccharides, but their substrate spectra are limited to the same family. Thus, the degradation activity across families of GAG lyases is advantageous and desirable for various applications. In this study, residue Lys130 at the substrate entrance of monomeric heparinase III from Pedobacter heparinus ATCC 13125 was replaced by cysteine, and the resulting mutant K130C showed novel catalytic activity in degrading hyaluronic acid without affecting its native activity toward heparin and heparan sulfate. The broadened catalytic promiscuity by mutant K130C was the result of dimerization through a disulfide bond to expand the substrate binding pocket. This bifunctional enzyme is potentially valuable in the degradation of different types of GAGs.

Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

“…Proteoglycans are composed of core proteins to which chains of anionic polysaccharides and glycosaminoglycans (GAGs) are attached. GAGs consist of repeated disaccharide units [ 22 , 70 ]. Depending on the composition of these units, GAGs are classified as heparin or heparan sulfate, dermatan sulfate, chondroitin sulfate, keratin sulfate and hyaluronic acid [ 16 , 70 ].…”

“…The best-characterized bacterial heparinases are the three enzymes produced by Pedobacter heparinus , (previously known as Flavobacterium heparinum ) heparinase I, II, and III [ 16 , 17 , 22 , 24 – 27 ]. No significant homology exists between either the DNA sequences of the genes that code for the three heparinases or the amino acid sequences of the proteins [ 28 ].…”

“…No significant homology exists between either the DNA sequences of the genes that code for the three heparinases or the amino acid sequences of the proteins [ 28 ]. The three heparinases proved to be valuable tools in analyzing different structural, biochemical and physiological properties of heparin and heparan sulfate-like glycosaminoglycans [ 18 , 22 , 24 , 26 ]. Additional uses of P. heparinus heparinases include heparinase I as a heparin antagonist [ 16 , 29 ] and heparinases I, II, and III to reduce neovascularization during tumor progression through their ability to alter the action of fibroblast growth factor [ 16 , 20 , 21 , 27 ].…”

Photoswitchable Heparinase III for Enzymatic Preparation of Low Molecular Weight Heparin

“…To investigate the precise control of enzymatic activity from the irradiation by a blue light and UV-LED, the cleaved fragments from heparin by photosensitive K130C–DMAA conjugate were analyzed and compared. As shown in Figure B, in accordance with the cis conformation of DMAA, oligosaccharide products from heparin by UV-irradiation (365 nm, 5 mW/cm 2 , 5 cm) of K130C–DMAA conjugate for 60 min were detected by DNS chromogenic method, which is comparable to the products produced by wild-type Ph HepIII. The activity of K130C–DMAA conjugate in the active cis form for the enzymatic deploymerization of heparin (0.75 μg/min/mg) was similar to that of wild-type HepIII (0.8 μg/min/mg) and mutant K130C (0.78 μg/min/mg).…”

“…According to the crystal structure of heparinase III from Pedobacter heparinus ( Ph HepIII), the site of Lys130 is adjacent to the active pocket and presented at the surface of the substrate entrance. Meanwhile, regardless of its structure of being a monomer or disulfide-linked homodimer, mutant K130C of Ph HepIII was previously demonstrated to completely retain the activity on heparin degradation from the comparison of various mutants around the active site, which provides a unique opportunity for site-specific coupling of the only cysteine residue with an azobenezene moiety for the regulation of its degradation activity to prepare LMWH.…”