کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1929305 | 1050452 | 2012 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli](/preview/png/1929305.png)
We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and β-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328–2 (ReNHase) was expressed with (ReNHase+Act) and without (ReNHase−Act) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4 Å resolution revealing an αβ heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.
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► Functional expression of an Fe-type nitrile hydratase without an activator protein.
► X-ray structure of the nitrile hydratase from Comamonas testosteroni Ni1 at 2.4 Å.
► Iron active site resides at the bottom of a wide solvent exposed channel.
► Two His residues reside in the active site that are not conserved.
► Solvent exposed active site and/or His residues may play a role in iron incorporation.
Journal: Biochemical and Biophysical Research Communications - Volume 424, Issue 3, 3 August 2012, Pages 365–370