A novel in vivo biosynthesis approach for split variants and diagnostic application of Rhodobacter capsulatus xanthine dehydrogenase

【Author】

Cheng-Hua Wang;Chong Zhang;Xin-Hui Xing;

【Abstract】

As a valuable oxidoreductase that catalyzes the oxidative metabolism of purines and aldehydes on CH or CH2 groups using NAD(P)~+as the electron acceptor,xanthine dehydrogenase(EC 1.17.1.4,XDH)can be used in a wide range of applications,such as diagnosis of clinical diseases,the synthesis of nucleoside drugs and the detection and degradation of organic pollutants.The production of commercial XDHs represented by bovine(αβγ)_2 XDH usually includes a post-translational proteolysis to increase its catalytic activity.However,the proteolysis approach has the low controllability,low efficiency,and difficulty for the purification process.In an attempt to obviate these disadvantages,this study proposed a novel method to in vivo biosynthesize split XDHs,and a proof of concept test was carried out on a well-studied model XDH,Rhodobacter capsulatus XDH,by translating novel active(αβγ)_2 XDH through directly expressing the iron-sulfur clusters([2Fe-2S]),flavin adenine dinucleotide(FAD)and sulfurated molybdenum pterin(Moco)domains as three separate redox proteins in Escherichia coli.Two novel(a(3y)2 XDH variants,Splitl66 and Splitl78,that were designed by splitting the small subunit of R.capsulatus CGMCC 1.3366(ap^XDH at the N-and C-terminal ends of the Li67-Ai78 peptide linking the iron-sulfur clusters and flavin adenine dinucleotide domains,respectively,were expressed in E.coli and characterized.SDS-PAGE and native-PAGE results suggested that both the split variants self-assembled into active(αβγ)_2 form XDHs.All the split variants and wild type XDHs contained the three redox centers as demonstrated by the UV-Vis,ICP-MS and reverse phase HPLC analyses,although their contents are different.Far-UV CD spectra indicated the very similar secondary structural composition(SSC)of the split variants and wild type XDHs.Compared to the wild type,both the split variants increased the thermostability by 11℃,and Split 178enhanced the turnover number and catalytic efficiency by 1.15-fold and 1.65-fold,while Splitl66 decreased by 3.2-fold and 5-fold,respectively.Computational modeling suggested the enzymatic property changes by splitting might be attributed to the domain structures,interdomain interactions and the redox centerrearrangement.This study was the first successful trail to express an active split(αβγ)_2 XDH directly by manipulating genes encoding redox domains,and the enhanced properties of the expressed(αβγ)_2 XDH by the in vivo splitting strategy might be a promising way for the commercial production of XDHs.As a demo application,the recombinant R.capsulatus XDH in this study showed a linear relationship between the measured and actual values of the xanthine content in both the blood and urine fluidics within the range of0~0.4 mM(R~2=0.999),outperforming the commercial Sigmal test kits,which employed bovine milk(αβγ)_2 XDH and diagnosed accurately within the range of 0~0.24 mM xanthine in fluidics(R~2=0.90).

【Keywords】

Genetic engineering;;Molecular modeling;;Purine metabolism;;Proteolysis;;Redox center;;Rhodobacter capsulatus;;Split protein;;Xanthine dehydrogenase

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