AnhE, a metallochaperone involved in the maturation of a cobalt-dependent nitrile hydratase

Sachi Okamoto, Filip Van Petegem, Marianna A. Patrauchan, Lindsay D. Eltis

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30 Citations (Scopus)


Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 μMCoCl2. Ten- to 100-fold less CoCl 2 sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mM phosphate, pH 7.0, 25 °C). Divalent metal ions including Co2+, Cu2+, Zn2+, and Ni2+ stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co2+ with Kd of 0.12 ± 0.06 nM and 110 ± 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn2+ (Kd = 11 ± 2 nM) and Ni2+ (Kd = 49 ± 17 nM) and did not detectably bind Cu2+. Substitution of the sole histidine residue did not affect Co2+ binding. Holo-AnhE had a weak absorption band at 490nm(∈ = 9.7 ± 0.1M-1 cm-1), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.

Original languageEnglish
Pages (from-to)25126-25133
Number of pages8
JournalJournal of Biological Chemistry
Issue number33
Publication statusPublished - Aug 13 2010
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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