کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
8305691 1538431 2014 11 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T
چکیده انگلیسی
Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent Tm of 62 ± 1 °C. Optimal parameters for the enzyme activity were pH 7.5 and 50 °C, showing apparent Km and kcat values of 17.7 ± 2.7 mM and 24.4 ± 1.5 s−1, respectively. Co2+ proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3-4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H2O2 toward allantoinase.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimie - Volume 99, April 2014, Pages 178-188
نویسندگان
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