Mutational and structural analysis of cobalt‐containing nitrile hydratase on substrate and metal binding

Abstract

Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K m and K i values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of bTyr68 formed hydrogen bonds with both n-butyric acid and aSer112, which is located in the active center. The bY68F mutant showed an elevated K m value and a significantly decreased k cat value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between aCys108 and aCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (aT109S and aY114T) were constructed, each residue being replaced with an iron-containing one. The aT109S mutant showed similar characteristics to the wild-type enzyme. However, the aY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of aCys111 and aCys113 residues were not observed. The aTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

“…The crystal structure of the apo-NHase in Pseudonocardia thermophila JCM3059 has shown that the oxidation of cysteine residues in the active center does not occur, but electron density connecting Cys-108 and Cys-113 of the ␣-subunit was observed, indicating the formation of a disulfide bond between the two residues (25). Considering this finding, we speculate that a disulfide bond should also exist between the corresponding Cys-109 and Cys-114 in the ␣-subunit of apo-␣e 2 and that the cobalt incorporation would be blocked by the disulfide bond.…”

“…The post-translationally oxidized Cys-SO 2 H and Cys-SOH have deprotonated Cys-SO 2 Ϫ and Cys-SO Ϫ structures, respectively (3), and the deprotonated Cys-SO 2 Ϫ and Cys-SO Ϫ in the holo-␣-subunit form salt bridges with two arginines of the ␤-subunit (which are conserved in all known Co-type and Fe-type NHases) in the holoenzyme (8 -11). The apoenzyme is likely to be non-oxidized, judging from the results of previous studies on NHase (25) and the related enzyme thiocyanate hydrolase (26 -28). Such cysteine oxidation has been demonstrated to occur in cobalt-containing NhlAE (holo-␣e 2 ), but not in cobalt-free NhlAE (apo-␣e 2 ), and the cysteine oxidation plays an essential role in self-subunit swapping maturation (20).…”

Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

“…[1][2][3] The active site can contain either a low-spin Fe III or a lowspin Co III ion coordinated by three cysteines and two deprotonated amides (two amides and two cysteines occupying the equatorial plane with an axial cysteine). [5][6][7] The Fe enzyme is isolated in a diamagnetic NO-bound form, activated by irradiation, and is proposed to be stabilized by the presence of butyric acid in the medium. 4 The sixth coordination site is occupied by a NO (as isolated) or an H x O molecule (after photolysis).…”

“…11,12 The catalytic mechanism of this site is not well understood, and there are several proposals regarding the role of the low-spin Fe III center in activating nitriles for hydrolysis. 13 The possible functional role of these modified cysteines, and the electronic structure of the asisolated [FeNO] 6 species (using the Enemark-Feltham nomenclature, where the superscript 6 refers to the total number of valence electrons on the iron and the π* orbitals of NO) and its observed photochemistry, have been a focus of several recent experimental and theoretical studies. [14][15][16][17][18] A significant number of structurally relevant model complexes have been reported, some of which bind nitriles and some of which reversibly bind NO and show photolytic behavior.…”

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

“…N-771. [38][39][40] These observed shifts have been attributed to a change in the π-back-donation from the axial cysteine ligand to the metal center, in response to perturbations at the active site. 41,42 Fig.…”