Heterologous expression and characterization of the ene-reductases from Deinococcus radiodurans and Ralstonia metallidurans
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- 作者：S. Litthauer ; S. Gargiulo ; E. van Heerden ; F. Hollmann ; D.J. Opperman
- 关键词：Old Yellow Enzyme ; Ene-reductase ; 伪 ; 尾-Unsaturated carbonyl ; Cofactor regeneration ; Flavin oxidoreductase
- 刊名：Journal of Molecular Catalysis B: Enzymatic
- 出版年：January, 2014
- 年：2014
- 卷：99
- 期：Complete
- 页码：89-95
- 全文大小：1977 K
文摘
The Old Yellow Enzyme (OYE) homologues or ene-reductases (ER) from Deinococcus radiodurans (DrER) and Ralstonia metallidurans (RmER) were cloned and characterized. Sequence and phylogenetic analysis revealed both these enzymes to belong to the YqjM-like or 鈥渢hermophilic-like鈥?group of OYEs, both sharing more than 60% sequence similarity to the ER from Thermus scotoductus. This group of OYEs is characterized by a conserved cysteine residue modulating the redox potential of the flavin cofactor as well as a conserved tyrosine residue located at the N-terminus region involved in binding certain ligands. The genes were recombinantly expressed in Escherichia coli as functional soluble proteins. Both ERs have monomer molecular weights of approximately 40 kDa, with DrER a homodimer in solution and RmER a monomer. DrER and RmER are optimally active at pH 7-7.5 at 30 掳C and 35 掳C respectively. Although the enzymes showed comparable affinities towards the ubiquitous ER substrate 2-cyclohexenone, the specific activity and catalytic efficiency of DrER were more than twice those observed for RmER. Similar to other members of this subclass of ERs, no conversion was detected with cyclic C尾 substituted enones, and only DrER was able to convert citral. Both DrER and RmER were highly active at reducing N-phenyl substituted maleimides. The selectivity of the ERs was assessed using both the isomers of carvone, which were converted with high diastereomeric excesses. Ketoisophorone and 2-methylcyclopentenone were converted to their (R)- and (S)-enantiomeric products respectively. Finally, a light-driven cofactor regeneration system was used to drive enzymatic reduction in the absence of NAD(P)H.