Abstract
The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at −20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia.
Highlights
Uricase or urate oxidase (EC 1.7.3.3; UOX) is a cofactor-free homotetrameric enzyme that catalyses the conversion of uric acid to 5-hydroxyisourate, an unstable intermediate that can undergo spontaneous or enzymatic hydrolysis to allantoin
Allopurinol, a member of xanthine oxidase inhibitors, is the most widely prescribed. Usage of these drugs is not without problems; uricosuric agents are ineffective if renal function is impaired, allopurinol can induce allergic reactions and severe hypersensitivity [8] and refractory gout was reported to associate with patients receiving allopurinol [9]. These agents reduce serum uric acid (SUA) level very slowly, the drugs are inappropriate for treatment of acute hyperuricemia due to tumour lysis syndrome (TLS), which usually presents with very high level of SUA
Two forms of clinically approved UOXs are available in the market; recombinant Aspergillus flavus UOX, which is approved for TLS [15] and PEGylated porcine-baboon chimera (PBC) UOX, which is approved for chronic refractory gout [16]
Summary
Uricase or urate oxidase (EC 1.7.3.3; UOX) is a cofactor-free homotetrameric enzyme that catalyses the conversion of uric acid to 5-hydroxyisourate, an unstable intermediate that can undergo spontaneous or enzymatic hydrolysis to allantoin. Allopurinol, a member of xanthine oxidase inhibitors, is the most widely prescribed Usage of these drugs is not without problems; uricosuric agents are ineffective if renal function is impaired, allopurinol can induce allergic reactions and severe hypersensitivity [8] and refractory gout was reported to associate with patients receiving allopurinol [9]. These agents reduce SUA level very slowly, the drugs are inappropriate for treatment of acute hyperuricemia due to tumour lysis syndrome (TLS), which usually presents with very high level of SUA. The LM-UOX has potential to be developed for treatment of hyperuricemia
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