Abstract
Urate oxidase is a key enzyme in purine metabolism and catalyzes the oxidation of uric acid to allantoin. It is used to treat hyperuricemia and gout, and also in a diagnostic kit. In this study, error-prone polymerase chain reaction and staggered extension process was used to generate a mutant urate oxidase with improved enzyme activity from Bacillus subtilis. After several rounds of mutagenesis and screening, two mutants 6E9 and 8E279 were obtained which exhibited 2.99 and 3.43 times higher catalytic efficiency, respectively. They also exhibited lower optimal reaction temperature and higher thermo-stability. D44V, Q268R and K285Q were identified as the three most beneficial amino acid substitutions introduced by site-directed mutagenesis. D44V/Q268R, which was obtained through random combination of the three mutants, displayed the highest catalytic activity. The Km, kcat/Km and enzyme activity of D44V/Q268R increased by 68%, 83% and 129% respectively, compared with that of wild-type urate oxidase. Structural modeling indicated that mutations far from the active site can have significant effects on activity. For many of them, the underlying mechanisms are still difficult to explain from the static structural model. We also compared the effects of the same set of single point mutations on the wild type and on the final mutant. The results indicate strong effects of epistasis, which may imply that the mutations affect catalysis through influences on protein dynamics besides equilibrium structures.
Highlights
Urate oxidase, which is known as uricase, is an important enzyme in purine degradation
In order to improve the activity of wild type Bacillus subtilis urate oxidase (BSUO), random mutagenesis libraries were constructed through error prone PCR (EP-PCR)
There is a high level of uric acid in human body which reduces the level of free radicals and the possibility of cancer [4, 21]
Summary
Urate oxidase, which is known as uricase, is an important enzyme in purine degradation. It catalyzes uric acid degradation and produces allantoin (Fig 1), which is much more soluble than uric acid [1]. During the evolutionary process, urate oxidase activity seems to have been lost in some higher primates including humans [3, 4]. In these species, the end product of purine metabolism is uric acid rather than allantoin [1].
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