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The Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1 Does Not Require an Activator Accessory Protein for Expression in Escherichia coli
Biochemical and Biophysical Research Communications
  • Misty L. Kuhn, Loyola University Chicago
  • Salette Martinez, Loyola University Chicago
  • Natalie Gumataotao, Loyola University Chicago
  • Uwe Bornscheuer, Greifswald University
  • Dali Liu, Loyola University Chicago
  • Richard C. Holz, Marquette University
Document Type
Article
Publication Date
8-3-2012
Disciplines
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 2.4 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.

Comments

Biochemical and Biophysical Research Communications. Vol. 424, No. 3 (August 3, 2012): 365-370. DOI.

Richard C. Holz was affiliated with Loyola University-Chicago at the time of publication.

Citation Information
Misty L. Kuhn, Salette Martinez, Natalie Gumataotao, Uwe Bornscheuer, et al.. "The Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1 Does Not Require an Activator Accessory Protein for Expression in Escherichia coli" Biochemical and Biophysical Research Communications (2012) ISSN: 0006-291X
Available at: http://works.bepress.com/richard_holz/4/