Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 424, Issue 3, Pages 365-370Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2012.06.036
Keywords
Nitrile hydratase; Iron; Hydrolysis; X-ray crystallography
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Funding
- National Science Foundation [CHE-1058357]
- ACS Petroleum Research Fund [ACS PRF 50033-ND4]
- Direct For Mathematical & Physical Scien
- Division Of Chemistry [1058396] Funding Source: National Science Foundation
- Direct For Mathematical & Physical Scien
- Division Of Chemistry [1435915] Funding Source: National Science Foundation
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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 alpha- and beta-subunit genes of the Fe-type NHase Comamonas testosteroni Nil (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 angstrom resolution revealing an alpha beta 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. (C) 2012 Elsevier Inc. All rights reserved.
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