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Biochem Biophys Res Commun. 2012 Aug 3;424(3):365-70. doi: 10.1016/j.bbrc.2012.06.036. Epub 2012 Jun 16.

The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli.

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1
Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W. Sheridan Rd., Chicago, IL 60626, United States.

Abstract

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 2.4Å 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.

PMID:
22713452
DOI:
10.1016/j.bbrc.2012.06.036
[Indexed for MEDLINE]
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