Enhanced catalytic properties of novel (αbγ)2 heterohexameric Rhodobacter capsulatus xanthine dehydrogenase by separate expression of the redox domains in Escherichia coli

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Post-translational proteolysis is usually necessary for the commercial production of xanthine dehydrogenases (XDHs), such as bovine (αβγ)2 XDH, to increase its catalytic activity. The proteolysis approach suffers from low controllability and inefficient purification. To obviate these disadvantages, we have developed a method that translates active Rhodobacter capsulatus (αβγ)2 XDH by directly expressing the iron-sulfur domain, the flavin adenine dinucleotide domain and the sulfurated molybdenum domain as three separate proteins in Escherichia coli. Two (αβγ)2 XDH variants, Split166 and Split178, which were designed by splitting the small subunit of R. capsulatus CGMCC 1.3366 (αβ)2 XDH at the N- and C-terminal ends of the L167-A178 peptide linking the iron–sulfur clusters and flavin adenine dinucleotide domains, respectively, were expressed in E. coli. Compared to the wild type, both split variants increased the thermostability by 11°C and Split178 enhanced the turnover number and catalytic efficiency by 1.15-fold and 1.66-fold, while Split166 decreased these parameters by 3.2-fold and 5-fold, respectively. This study is the first successful trial 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 using the in vivo splitting strategy may be a promising technique for the commercial production of XDHs.