UBC Theses and Dissertations
Characterization of a novel cobaltoenzyme and its metallochaperone in rhodococcus Okamoto, Sachi
Rhodococcus jostii RHA1 grows on various nitriles. Proteomic studies revealed that RHA1 utilizes an nth pathway, which includes an Fe³⁺-dependent nitrile hydratase (NHase), to catabolize phenylacetonitrile. By contrast, RHA1 utilizes the anh pathway, which includes a novel acetonitrile hydratase (ANHase), a αβ-heterodimeric metalloenzyme, to catabolize acetonitrile. To better characterize ANHase, a Rhodococcus-E. coli shuttle vector was constructed using a benzoate-inducible promoter. ANHase transformed nitriles containing up to four carbons, with highest specificity for acetonitrile and propionitrile. The enzyme contained cobalt, copper and zinc, and lacked sequence identity with known NHases. Accordingly, ANHase is proposed to belong to a previously unknown class of NHases. The α subunit of ANHase possesses two potential metal-binding sequences: an N-terminal sequence rich in His and acidic residues; and a Cys-rich motif (CLLGCAC). The latter is reminiscent of the conserved catalytic motif in Co³⁺-dependent NHases, suggesting that it coordinates Co³⁺ in ANHase. The N-terminal sequence is similar to that found in some other cobaltoenzymes. Spectrophotometric analysis of a synthetic N-terminal peptide (MPDHGHDHGHNHDACDSE) demonstrated that it formed a 2:1 complex with Co²⁺ and that Cys is a probable ligand. Isothermal titration calorimetry (ITC) studies demonstrated that the peptide binds Co²⁺, Zn²⁺ and Ni²⁺ with similar affinities (Kd = 3.2–4.3 µM). Finally, AnhE, an 11-kDa protein whose gene occurs between the ANHase structural genes, anhA and anhB, was characterized. A ΔanhE mutant grew on acetonitrile only if anhE was provided in trans. Co-expression of anhE with anhA enabled reconstitution of ANHase in vitro in the presence of low concentrations of Co²⁺. Co²⁺ and other divalent metal ions stabilized a dimeric form of AnhE. ITC studies demonstrated that the dimer binds two Co²⁺ ions with different affinities (Kd1 = 0.12 nM, Kd2 = 110 nM) but only one Zn²⁺ (Kd = 11 nM) and one Ni²⁺ (Kd = 49 nM). Together, these data suggest that AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. Overall, these studies provide insight into a novel NHase and its maturation. The findings have potential applications in the bacterial transformation of nitriles as well as implications for metal-trafficking in biological systems.
Item Citations and Data
Attribution-NonCommercial-NoDerivatives 4.0 International