کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1926939 1536491 2007 13 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A shuffled CYP2C library with a high degree of structural integrity and functional versatility
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
A shuffled CYP2C library with a high degree of structural integrity and functional versatility
چکیده انگلیسی

Cytochrome P450 (CYP) enzymes involved in mammalian xenobiotic metabolism are attractive targets for the engineering of biocatalysts since they have broad and overlapping substrate and reaction substrate specificities. In this report, a library of chimeric mutants was prepared from CYP2C8, CYP2C9, CYP2C18 and CYP2C19 by DNA family shuffling. Twelve randomly selected clones were fully sequenced and showed 9 ± 2 crossovers and 1.5 ± 0.5 spontaneous mutations per ∼1.5 kbp open reading frame. CYP hemoprotein expression was observed in 50% (microaerobic culture) to 54% (aerobic culture) of clones. The functional diversity of the library was assessed using three luminogenic substrates, diclofenac and indole as probe substrates. A random sample of 26 clones revealed two clones with activity towards luciferin ME, one towards luciferin H and five towards diclofenac 4′-hydroxylation. One mutant showed activity towards all three substrates. Of 96 clones screened on solid media, one showed elevated indigo production compared to the parental forms. Turnover rates for luciferin ME and H metabolism by CYP2C9 and mutants were at least one order of magnitude higher in experiments with membranes compared to whole cells, consistent with impaired product egress from cells. Apparent Km values were increased in whole cell incubations with luciferin H suggesting impaired access of the substrate to the active site of the enzymes in whole cells. Finally screening with a panel of CYP2C ligands using CYP2C9 or active mutants revealed different patterns of inhibition and heteroactivation of metabolism of luciferin analogs.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 467, Issue 2, 15 November 2007, Pages 193–205
نویسندگان
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